LOW FREQUENCY FATIGUE IN ENDURANCE TRAINED, …

LOW FREQUENCY FATIGUE IN ENDURANCE TRAINED, SEDENTARY,

AND SPINAL CORD INJURED SUBJECTS

by

EDWARD THOMAS MAHONEY

(Under the direction of Dr. Kevin McCully)

ABSTRACT

This study examined low frequency fatigue (LFF) in endurance trained (ET) and

sedentary (SED) able-bodied subjects, and in individuals with spinal cord injury (SCI). ET and SED performed two separate neuromuscular electrical stimulation (NMES) protocols to evoke fatigue of the quadriceps of one thigh (experimental leg) with the un-fatigued leg as a control. Protocol 1 (‘15 Min’) lasted 15 min and the duty cycle was 33%. For protocol 2, fatigue in SED was matched to ET during the 15 min protocol (‘Low Matched’) while fatigue in ET was matched to the SED 15 min protocol (‘High Matched’). Force was assessed at 20 Hz (P20) and 100 Hz (P100) and the ratio of P20/P100 was used to evaluate LFF in both thighs before and up to 24 hours following fatigue. The SCI group performed only one protocol in which fatigue was matched to SED during the 15 min protocol, and evaluation of LFF was the same. Results indicated that SED had a greater magnitude of LFF compared to ET with the 15 Min (p<0.001) and High Matched (p<0.020) comparisons. The ET group did not recover faster than the SED group for any of the comparisons. Muscle pain 24 hours after the fatigue tests may have affected LFF values. For SCI, the magnitude of LFF was not significantly different compared to SED. Recovery of LFF was faster in SED compared to SCI in both the experimental (p<0.001) and control leg (p<0.001). SCI did not recover from LFF over 24 hours in either leg. When LFF values in the experimental leg were corrected for LFF values in the control leg, no difference in recovery existed between SED and SCI (p=0.064). In summary, ET had less LFF than SED even when total fatigue was matched, suggesting that ET muscle is more ‘protected’ from LFF. Although ET did not recover faster from LFF, other factors such as muscle injury may make interpretations of recovery difficult. When paralyzed muscle is stimulated sufficiently, LFF will be substantial for at least 24 hours. In addition, assessing LFF with NMES in SCI causes a progressive increase in LFF, which is likely due to muscle injury. INDEX WORDS: Low frequency fatigue, electrical stimulation, spinal cord injury, endurance training, calcium impairment.



by


B.S. Ithaca College, 1995

M.A. University of Georgia, 1998

A Dissertation Submitted to the Graduate Faculty of The University of Georgia in

Partial Fulfillment of the Requirements for the Degree

DOCTOR OF PHILOSOPHY

ATHENS, GEORGIA

2006



by


Major Professor: Kevin McCully

Committee: Gary A. Dudley Rod Dishman

Kirk J. Cureton Patrick L. Jacobs

Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia August 2006

DEDICATION

I dedicate this work to my wife, Melanie who has always been supportive and is my pillar

of strength. Melanie, I love you and I am proud to be your husband.

Also, I dedicate this to Dr. Gary A. Dudley and his family as they have all faced extreme

challenges and hardships over the past 4 years. When most individuals would have given up due

to these overwhelming circumstances, Dr. Dudley continues to be dedicated to his work, and

more importantly, his family. He has the heart of a lion and an unending drive to acquire

knowledge and achieve excellence. He is a true inspiration to me, and to all that know him.

iv

ACKNOWLEDGMENTS

I would like to thank Dr. Kevin McCully for all the time you spent with me on this

project and for your guidance and support throughout my doctoral degree. I have enjoyed

working with you very much and hope that we might collaborate in the future. Dr. Gary Dudley,

thank you for giving me the opportunity to work in your lab and to learn from you over the past 4

years. You are a true inspiration to all and your scientific knowledge is second-to-none. Dr. Kirk

Cureton and Dr. Rod Dishman, thank you for your input and guidance on this project and for all

the valuable information I learned in your classes. To my former mentor, Dr. Patrick Jacobs,

thank you for your guidance on this project. More importantly, thank you for all the time you

spent with me when I first started learning about individuals with SCI. You are the first person

who sparked my interest in SCI research and I am grateful for all that I learned from you over the

past years. Also, I would like to thank Dr. Debbie Backus at Shepherd Center for facilitating my

data collection of SCI subjects and for all her help related to this project.

I would like to thank Mrs. Dudley for her amazing strength over the past 4 years. You

and your family will always be in our prayers. Also, our lab, and practically the entire building,

would like to thank you for the almost unending supply of snacks to fuel our brains and add mass

to our bodies! I would like to thank Timmy ‘Stat Master’ Puetz for all of his help with my

statistical analyses and for his friendship. Also, I would like to thank all my research subjects for

participating in my study. To the lab members, Chris Elder, Tracey ‘Tricky’ Kendall, and Chris

Black thanks for all the good times in the lab and for your help with this project. To my brother

v

Dave, I would like to thank you for your support and friendship over the past years and for all the

laughs we have had throughout our lives.

Lastly, I would like to thank my wife, Melanie for all her support and patience with me

over the past 4 years. I would have never made it without you. Thank you for keeping me

positive and for believing in me. I am excited to begin a new chapter of life with you in

beautiful Louisville, KY.

vi

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS.............................................................................................................v

LIST OF TABLES......................................................................................................................... ix

LIST OF FIGURES .........................................................................................................................x

CHAPTER

I. INTRODUCTION.........................................................................................................1

Purpose.....................................................................................................................4

Specific Aims...........................................................................................................4

Hypotheses...............................................................................................................4

Significance of the Study .........................................................................................5

Limitations of the Study...........................................................................................6

II. REVIEW OF LITERATURE.......................................................................................8

Muscle Fatigue.........................................................................................................8

Fatigue and Endurance Training..............................................................................8

Calcium Related Fatigue..........................................................................................9

Training Status and Low Frequency Fatigue (LFF) ..............................................12

Effect of Fiber Type and Metabolic Fatigue on LFF.............................................13

Muscle Injury and LFF ..........................................................................................14

Surface Neuromuscular Electrical Stimulation (NMES).......................................16

Muscle Fatigue and Spinal Cord Injury (SCI) .......................................................17

vii

Low Frequency Fatigue and SCI ...........................................................................17

References..............................................................................................................19

III. LOW FREQUENCY FATIGUE AFTER ELECTRICALLY EVOKED

CONTRACTIONS IN TRAINED AND UNTRAINED SUBJECTS.............…….25

Abstract ..................................................................................................................26

Introduction............................................................................................................27

Methods..................................................................................................................28

Results....................................................................................................................35

Discussion..............................................................................................................39

References..............................................................................................................44

IV. LOW FREQUENCY FATIGUE IN INDIVIDUALS WITH SPINAL CORD

INJURY .................................................................................................................55

Abstract ..................................................................................................................57

Introduction............................................................................................................58

Methods..................................................................................................................60

Results....................................................................................................................66

Discussion..............................................................................................................70

References..............................................................................................................76

V. SUMMARY AND CONCLUSIONS..........................................................................88

References..............................................................................................................91

viii

LIST OF TABLES

Page

Table 3.1: Subjects characteristics.................................................................................................48

Table 3.2: Muscle pain ratings in the experimental leg in endurance trained and sedentary

subjects 24 and 48 hours after completion of fatigue tests...........................................55

Table 4.1: Individual and mean data for spinal cord injured participants .....................................80

Table 4.2: Mean subject characteristics for SCI and able-bodied groups......................................81

ix

LIST OF FIGURES

Page

Figure 3.1: Percent reduction in force-time integrals during fatigue protocols in endurance

trained and sedentary subjects.....................................................................................49

Figure 3.2: Representative force tracings at 20 Hz and 100 Hz for one sedentary participant pre-

and 1-hour post-fatigue ...............................................................................................50

Figure 3.3: 100 Hz force values (% initial) for 15 Min (a), High Matched (b) and Low Matched

(c) conditions in ET and SED. ....................................................................................51

Figure 3.4: Control leg LFF values over 24 hours post-fatigue in ET and SED for the 15 Min

condition .....................................................................................................................52

Figure 3.5: Magnitude of LFF for 15 Min (a), High Matched (b) and Low Matched (c) conditions

in ET and SED over 1-hour post-fatigue.....................................................................53

Figure 3.6: Recovery of LFF for 15 Min (a), High Matched (b) and Low Matched (c) conditions

in ET and SED over 24 hours post-fatigue.................................................................54

Figure 4.1: Percent reduction in force-time integrals during fatigue protocols in spinal cord

injured and able-bodied subjects.................................................................................82

Figure 4.2: Representative force tracings at 20 Hz and 100 Hz for one spinal cord injured

participant pre- and 1-hour post-fatigue. ....................................................................83

Figure 4.3: 100 Hz force values (% initial) after a fatigue test for the experimental (a) and control

(b) leg in SCI and able-bodied over 24 hours post-fatigue .........................................84

x

Figure 4.4. Magnitude (a) and recovery of LFF (b) in the experimental leg of SCI and able-

bodied subjects. ...........................................................................................................85

Figure 4.5. Recovery of LFF in the control leg over the 24-hour post-fatigue period in SCI and

able-bodied subjects ....................................................................................................86

Figure 4.6. Recovery of LFF in the experimental leg when statistically adjusted for LFF in the

control leg of SCI and able-bodied subjects................................................................87

Figure 5.1. The mechanisms responsible for force loss along the Injury/Fatigue continuum.......93

xi

CHAPTER I

INTRODUCTION

Extensive literature exists regarding muscle fatigue (6, 21, 44, 45, 48, 59). Although

muscular fatigue has been examined following different modes and intensities of exercise in

many different subject populations, very small proportions of these studies have examined the

mechanisms involved in the fatigue. For example, many studies have reported that fatigue is

greater in individuals with spinal cord compared to able-bodied subjects, but few studies have

tried to determine what factors are responsible for the enhanced fatigue in those with paralysis.

Although the most widely known mechanisms of fatigue are usually thought to be

metabolic in nature, the potential role that calcium may play in fatigue cannot be disregarded.

The uptake and release of calcium from the sarcoplasmic reticulum (SR) are important

contributors to producing and maintaining force (2, 3, 19). Many studies have demonstrated that

muscular force can be reduced for several hours and may be related to disruptions in excitation-

contraction coupling (19, 72-74). However, studies directly measuring calcium levels with

muscular fatigue are typically invasive and are usually performed in animal models.

In humans, it is possible to approximate the impairment of calcium release following

muscular activity by examining low frequency fatigue (LFF), a well-documented phenomenon

(17, 62, 67). Low frequency fatigue is defined as a preferential loss of force at low stimulation

frequencies (ie. 20 Hz) compared to high frequencies (ie. 100 Hz). Low frequency fatigue is

commonly assessed by the ratio of force produced at low and high activation frequencies

following exercise (63, 66). It is characterized by a slow rate of force recovery and persistence of

1

reduced force in the absence of electrical or metabolic disturbances and has been shown to last 6-

8 hours or longer (17, 31, 63).

It is well established that aerobic athletes fatigue less during contractions than their

sedentary (SED) counterparts (27, 42, 44, 68). Despite this, few studies have measured the

various mechanisms related to the differences in fatigue between these two groups. However,

studies have shown that endurance trained (ET) athletes recover faster from metabolic fatigue

because their muscles are better equipped to buffer hydrogen ions and resynthesize

phosphocreatine and ATP levels (27, 44) due to high muscle oxidative capacity. However, the

question of how ET affects the long lasting recovery of muscular force (ie. LFF) due to

impairment in excitation-contractions coupling has not been fully examined.

Low frequency fatigue has been reported to be most dramatic in fast-twitch muscle fibers

(51, 53), as well as when prior muscle injury has occurred (13, 34, 46, 53). Highly trained

endurance athletes display exceptional muscular endurance and typically have greater

percentages of slow-oxidative muscle fibers (56) when compared to SED. These slow-oxidative

muscle fibers may be less susceptible to contraction-induced muscle injury (22, 38). Also, it

appears that endurance training itself may provide some protection (18). If ET individuals, in fact

have greater percentages of slow-oxidative fibers, and are more protected from contraction–

induced muscle injury, then it is possible that they would be less prone to LFF.

Independent of muscle injury, force loss observed during a bout of muscular contractions

due to metabolic factors has been shown to affect LFF (42, 50). Pronounced fatigue causes large

increases in inorganic phosphate and hydrogen ions. It is hypothesized that high levels of

inorganic phosphate may be taken up into the SR, where it may precipitate with calcium (23).

2

The formation of calcium phosphate would lower free calcium concentrations in the SR, thus

reducing calcium release.

A study by McCully et al. (42) determined that protocols that elicit high levels of fatigue,

and therefore metabolic byproducts, can increase the magnitude of LFF observed during

recovery. With regard to ET and SED individuals, previous literature would indicate that when

these two groups perform identical protocols that SED would incur greater metabolic fatigue and

force loss. For these reasons, it seems plausible that SED individuals would have greater LFF

following contractions, compared to ET subjects.

Surface neuromuscular electrical stimulation (NMES) is commonly used in testing of

able-bodied subjects as well as in those with neuromuscular disorders to facilitate contraction of

weak or paralyzed skeletal muscle (5, 11, 39). Activating skeletal muscle for the purpose of

functional movements and exercise through NMES is challenging as skeletal muscle fatigue can

occur rapidly. The profound fatigue that occurs with this modality, is likely due to altered motor

unit recruitment patterns (20, 35, 36), as well as synchronous activation of a given motor neuron

pool (1, 52). It is well established that the use of NMES requires greater energy demand and

consequently causes more fatigue when compared to similar voluntary efforts (29, 35, 69).

Muscle fatigue has been reported to be greater in individuals with spinal cord injury

(SCI) when compared to able-bodied subjects (5, 10, 30, 48). The most widely supported

mechanism explaining the high levels of fatigue observed in SCI subjects has been attributed to

the increased proportion of fast-twitch fibers (8, 25). To our knowledge, few studies have

thoroughly examined LFF in individuals with SCI. Since there is a somewhat complete

transformation from slow to fast contractile machinery with chronic SCI, as well as an increased

3

risk of contraction-induced muscle injury (5), it seems plausible that paralyzed muscle would be

more susceptible to LFF than normally loaded muscle.

Purpose

The general aim of this study is to better understand the mechanisms of muscle fatigue, in

particular in individuals who are extremely fit, as well as in those who are severely

deconditioned. This will be accomplished by measuring the magnitude and recovery of ‘low

frequency fatigue’ resulting from two NMES-evoked fatigue protocols in endurance trained (ET)

and sedentary (SED) able-bodied subjects, as well as in individuals with spinal cord injury (SCI).

Low frequency fatigue (LFF) will be measured for up to 24 hours in each of these groups after

performing two protocols which induce different amounts of fatigue. This study will determine if

the total amount of force loss during a fatigue protocol affects the LFF response or whether

additional factors play a role. Also, this study will determine if use of a control leg (un-fatigued

leg) when examining LFF is warranted.

Specific Aims

1. In ET and SED subjects, the magnitude and recovery rates of LFF were evaluated in response

to separate NMES-evoked protocols designed to initiate two levels of fatigue.

2. In SCI and SED subjects, the magnitude and recovery rates of LFF were evaluated in response

to a separate NMES-evoked protocol designed to match force loss in both groups.

Hypotheses

1a. ET will have less magnitude and faster recovery of LFF compared to SED following the

performance of an identical fatigue protocol (15 Min).

4

1b. ET will have less magnitude and a faster recovery of LFF compared to SED following the

performance of fatigue protocols designed to match relative force loss (High and Low Matched

conditions).

2. SED able-bodied controls will have less magnitude and faster recovery of LFF compared to

SCI following the performance of fatigue protocols designed to match relative force loss

between groups.

Significance of the study

It is unknown if calcium related fatigue (ie. LFF) can be reduced with endurance training,

even though it is well known that overall muscle fatigue is greatly reduced. SCI is a condition

associated with enhanced muscle fatigue and increased susceptibility to contraction-induced

muscle injury, both of which have been shown to enhance LFF. The increased muscle

fatigue/injury in SCI limits the ability to use NMES as a therapeutic tool. This study is the first

to thoroughly examine LFF in subjects with SCI, as well as how training status influences the

LFF response. An important aspect of this study will be to monitor the magnitude and recovery

of LFF after a fatiguing bout of exercise. This study is unique in that it tested if enhanced fatigue

with deconditioning (SED and SCI) is related to a large magnitude and prolonged recovery of

LFF, and whether electrical stimulation protocols causing different levels of fatigue will affect

this response. Using various fatigue tests allowed examination of LFF after the same number of

stimulations and after the same percentage of force loss between groups.

By performing this study, more was learned about the importance of LFF in humans after

use of NMES. Low frequency fatigue has clinical implications in that individuals with SCI, or

other neuromuscular disorders, may use low-frequency NMES several times a day for

5

rehabilitation to make weak or paralyzed muscle contract. The results of this study may aid

clinicians in designing appropriate electrical stimulation protocols for rehabilitation.

Limitations of the study

This study assessed LFF, a phenomenon thought to be due to impaired calcium release

during muscular contractions. This study, however, did not directly measure calcium release

rates and was not designed to determine the role that various mechanisms may have on LFF (ie.

muscle injury that may occur). The ratio of force produced at low and high frequency (P20/P100)

was assessed after a fatigue protocol to quantify LFF. The magnitude and recovery of LFF may

give a rough idea of how much excitation-contraction coupling is impaired, but LFF data was not

used to generalize calcium uptake or release rates.

Another potential limitation of this study is that subjects had to re-enter the force chair

several times throughout the 24-hour post-fatigue period, which may lead to increased variability

in the force measurements. To limit variability of testing measures, a control leg was utilized in

the research design. In addition, subjects were asked to remain relatively inactive over the course

of the day and the electrodes were traced to ensure the same placement for the 24-hour

assessment of LFF, or at any other time the electrodes were taken off during testing.

It has been reported that LFF is more pronounced when prior muscle injury has occurred.

This is not a large concern for the able-bodied subjects as research has shown that these types of

contractions do not typically induce muscle injury. However, NMES-evoked isometric

contractions are capable of eliciting muscle injury in those with long-term SCI. Although we

tried to limit muscle injury by keeping force values low, we did not quantitatively assess muscle

injury, which could potentially limit the interpretation of the results of this study, especially in

6

the SCI group. However, if NMES-evoked force returns to baseline values 24 h post-fatigue,

then we can assume little, or no injury has occurred.

7

CHAPTER II

REVIEW OF LITERATURE Muscle fatigue Skeletal muscle fatigue is defined as the “failure to maintain the required or expected

power output” (16). Skeletal muscle fatigue ordinarily occurs due to a number of plausible

theories including failure at the neuromuscular junction, decreased calcium release and reuptake,

buildup of metabolic byproducts, and central fatigue (21). In addition, factors outside the motor

unit, including inadequate blood flow, may contribute to muscle fatigue. Force recovery

following fatigue is made up of three distinct components (45). With the first component,

muscular force recovers quickly (within seconds) and is likely related to disruption in electrical

excitability of the muscle membrane or buildup of K+ in the transverse tubules, possibly related

to the disruption of Na+-K+ adenosinetriphosphatase pump function. The second component is

due to the build-up of various metabolites produced during exercise, which recover somewhere

between 3-20 minutes. The third component of fatigue, which has been demonstrated to last up

to 6-8 h and potentially longer, is likely related to disruption of excitation-contraction coupling

(calcium impairment), and force loss is most severe when muscle actions are elicited at low

frequencies (ie.1-20 Hz).

Fatigue and endurance training

It is well established that endurance trained (ET) athletes fatigue less and recover quicker

than their sedentary (SED) counterparts (27, 42, 44, 68). Theriault et al. (68) examined resistance

to fatigue in the knee extensor muscles of active and ET individuals. Subjects performed 25

8

maximal voluntary isometric contractions, each lasting 10 s with 5 sec rest between contractions

and demonstrated a significant difference in the amount of fatigue between both groups. This

study demonstrates that ET fatigue less with intermittent-type exercise, which is likely due to

faster recovery between contractions. McCully et al. (44) examined muscle metabolism in

controls and long-distance runners using 31P magnetic resonance spectroscopy. Phosphocreatine

was measured during and following 5 min of repeated plantar flexion of the calf muscles. The

maximal rates of PCr resynthesis were nearly twice as fast for long-distance runners as compared

to controls (64.8 vs. 38.6 mmol/min/kg muscle). The differences shown here are consistent with

literature demonstrating that long-distance runners have faster recovery of metabolites than

controls, which is directly due to higher oxidative capacity. Extensive literature exists regarding

muscle fatigue related to the buildup of metabolic byproducts, namely inorganic phosphate and

hydrogen ions, and their mechanisms of action are quite well known (44, 45, 69-71).

Calcium related fatigue (LFF)

The proposed project will not examine metabolic fatigue but will be more focused on

examining the long-lasting fatigue related to calcium impairment. Although some researchers

believe that loss of muscular force is almost completely due to the accumulation of metabolic

byproducts, it is becoming clear that calcium impairment may play a larger role in fatigue than

previously thought. The uptake and release of calcium from the sarcoplasmic reticulum (SR) are

important contributors to producing and maintaining force. It has been demonstrated that the

onset of muscle fatigue may be related to the inability of the SR to adequately release and

sequester calcium (19). Also, accumulation of metabolic byproducts themselves might cause

reductions in calcium release from the SR (2). Insufficient delivery of calcium to the

myofilaments would lead to reduced force output and may be caused by impaired excitation-

9

contraction coupling, changes in SR calcium concentrations, or by transient modifications of the

SR calcium channel (19). The strongest evidence points to transient modification of the SR

calcium channel with fatigue, which reduces its opening probability and decreases calcium

release into the myoplasm. Data from animal models indicate that when intracellular calcium

[Ca+2]i concentration are elevated, general calcium homeostasis becomes impaired and calcium

release is subsequently reduced (14, 37, 74). Also, it has been reported that high levels of [Ca+2]i

are capable of activating proteases that lead to cellular damage. Therefore, reducing calcium

release with fatigue would favor a lower calcium environment (due to continued sequestration of

calcium), which may act to maintain cellular integrity by limiting calcium-mediated damage (9).

Pronounced fatigue causes large increases in inorganic phosphate and hydrogen ions. It is

hypothesized that high levels of inorganic phosphate may be taken up into the SR, where it may

precipitate with calcium (23). The formation of calcium phosphate would lower free calcium

concentrations in the SR, thus reducing calcium release. However, the ensuing impairment in

calcium release would preferentially affect muscular forces at low activation frequencies.

Higher frequencies, however, are capable of overcoming such impairments and can produce

saturating levels within the myoplasm, thus blunting reductions in muscle force. From such

studies, it is becoming increasingly clear that impaired calcium handling is an important

mechanism involved in muscular fatigue.

Studies directly measuring calcium (concentrations, release/reuptake rates, receptor and

channel activity, etc.) with muscular fatigue are typically invasive and are usually performed in

animal models. Measuring calcium handling in humans is not as simple. In humans, we can

obtain muscle biopsies to measure calcium release, uptake, and Ca+2-adenosinetriphosphatase

activity with various protocols. From force tracings, we can obtain crude estimates of calcium

10

kinetics by examining rise and relaxation times during contractions. In addition, it is possible to

approximate the magnitude of calcium release impairment following muscular activity by

examining low frequency fatigue (LFF), a well-documented phenomenon. Low frequency

fatigue is defined as a preferential loss of force at low stimulation frequencies (ie. 20 Hz)

compared to high frequencies (ie. 100 Hz). Low frequency fatigue is commonly assessed by the

ratio of force produced at low and high activation frequencies following muscular fatigue (63,

66, 67). It is characterized by a slow rate of force recovery and persistence of reduced force in

the absence of electrical or metabolic disturbances and has been shown to last 6-8 hours or

longer (17, 31, 63).

After a bout of fatiguing exercise, force produced at low and high electrical stimulation

frequencies should both be affected by metabolic fatigue (41) which is known to subside ~20

min following exercise (45). High frequency force is less affected by this calcium related fatigue,

whereas low frequency force appears to be preferentially suppressed. Low frequency fatigue is

likely due to impaired excitation-contraction coupling with evidence specifically pointing to

blunted calcium release from the SR at low stimulation frequencies (17, 45, 73). Chin et al. (15)

have reported that the reduction in calcium release with LFF is primarily due to an elevation in

the [Ca+2]i-time integral, which represents the cumulative increase in [Ca+2]i above resting levels

for series of tetanic contractions.

A common misconception is that LFF is only caused by low frequency evoked muscle

contractions. Studies have elicited LFF with voluntary contractions, (41, 52, 63) as well as with

both high (60-100 Hz) and low frequency (1-30 Hz) electrically induced contractions (32, 52,

74). However, Ratkevicius et al. (52) have demonstrated that electrically evoked contractions

actually cause more LFF than do voluntary contractions and longer contractions (30 sec) cause

11

more pronounced LFF, as compared to shorter ones (5 sec). Although high frequency fatigue

protocols will elicit LFF, there is some evidence that lower frequencies cause a more pronounced

LFF (7). In addition to longer individual muscle contractions, longer duration fatigue protocols

themselves have been shown to cause greater LFF (4). In summary, LFF is most pronounced

following longer individual contractions which are electrically evoked, and that are performed

within fatigue protocols that are of longer duration.

Training status and LFF

A question that has not been fully examined is how training status may influence LFF

after fatiguing bouts of exercise. Since LFF has been attributed to impaired calcium release from

the SR, it has been suggested that high intensity training may help to partially compensate for

this impairment. A study by Willems & Stauber (75) examined LFF in rat plantar flexors with 6

weeks of high-speed eccentric resistance training. They showed significantly smaller reductions

in the P20/P100 ratio in trained vs. control muscles after an electrically stimulated fatigue test.

Although no calcium related measures were performed in this study, they attributed the reduction

in LFF in the resistance-trained muscles to increase release of calcium from the SR. In support of

this, Ortenblad et al. (49) demonstrated that 5 weeks of sprint training in men led to an enhanced

rate of peak SR calcium release, whereas calcium sequestration function was not changed.

Although calcium release may be enhanced with high intensity trained (sprint and resistance

training), it is likely that muscle fiber types do not get ‘faster’. If anything, this type of training

may cause a fiber type shift from type IIb to IIa, which is known to occur when sedentary

individuals become active (68). Something inherent about this fiber type shift may cause

reductions in LFF as it has been reported that fast-fatigable fibers are more prone to LFF than

fast-fatigue resistant fibers (51). Although high intensity training appears to reduce LFF

12

following a bout of exercise, there is not much information on how endurance training affects

LFF.

Effects of fiber type and metabolic fatigue on LFF

Following muscular contractions, LFF is evident in both fast and slow twitch muscle

fibers of animals, as well as humans (33). However, it has been reported that fast-glycolytic

fibers demonstrate greater LFF following types of muscular contractions than do fast-oxidative

fibers (51, 53). Powers & Binder (51) reported that fatigue resistant motor units from cat flexor

digitorum muscles exhibited less pronounced LFF than fast-intermediate and fast-fatigable motor

units after a series of electrically stimulated contractions. Also, they demonstrated a significant

positive correlation (r = 0.611) between tetanic tension of individual motor units and the

magnitude of LFF, indicating the greatest LFF was observed in the largest motor units.

Force loss observed during a bout of muscular contractions due to metabolic factors has

been shown to affect LFF (42, 50). In the cat gastrocnemius muscle, Parikh and colleagues (50)

examined LFF after protocols of 2, 5, 10, 20 and 50 concentric contractions at optimal muscle

length. As expected, they demonstrated that force loss after 50 concentric contractions was

greater than the trials where fewer contractions were performed. They demonstrated that as the

number of the contractions in a protocol increased, force loss was greater, and LFF was more

pronounced after exercise.

McCully et al. (42) used an in vivo rat muscle model to examine the potential role that

metabolic byproducts might have on calcium related fatigue (ie. LFF). They examined muscular

force and metabolic byproducts during and following bouts of electrical stimulation. Two

stimulation protocols were used: 1) high intensity stimulation followed by medium intensity

stimulation (High Group), and 2) low intensity stimulation followed by medium intensity

13

stimulation (Low Group). Metabolic fatigue was based on concentrations of inorganic phosphate

and LFF was assessed as the relative decline of force at low compared to high stimulation

frequencies. During the initial stimulation period, the High Group had greater metabolic fatigue

and LFF compared to the Low Group. During the second stimulation and recovery period, the

High group demonstrated no difference in metabolic fatigue but had significantly greater LFF.

They determined that protocols that elicit high levels of fatigue, and therefore metabolic

byproducts, can increase the magnitude of LFF observed during recovery. With regard to ET and

SED individuals, previous literature would indicate that when these two groups perform identical

protocols that SED would incur greater metabolic fatigue and force loss. It is possible that

greater metabolic fatigue observed in SED subjects may lead to a greater magnitude of LFF

following contractions, compared to ET subjects.

Muscle injury and LFF

Low frequency fatigue has been reported to be greater when prior muscle injury has

occurred (13, 34, 46, 53). Since the origin of LFF has been attributed to disruption in the

excitation-contraction coupling process, studies have demonstrated that muscle injury can impair

calcium release/reuptake rates and affect muscular force, most predominately at low activation

frequencies (9, 46). Rijkelijkhuizen et al. (53) examined the effects of 40 isometric, concentric,

or eccentric muscle actions on LFF in different fibers types of the rat medial gastrocnemius.

Seventy minutes post-exercise, LFF was more severe with eccentric compared with isometric

and concentric contractions. Of the three contraction types, eccentric actions cause the greatest

disruption to muscle fibers (43, 47). This study indicates that more injurious bouts of exercise

demonstrate greater LFF. Newham et al. (46) used a bench step test to initiate muscle injury

using concentric contractions in the quadriceps of one leg (step up leg) and eccentric contractions

14

in the other (step down leg). Regardless of contraction type, this protocol caused pronounced

LFF, indicated by a reduction in the P10/P50 ratio, which was still significantly suppressed 24

hours following bench stepping. However, the leg that performed the eccentric contractions had

a significantly larger reduction in P10/P50 as compared to the concentric leg, for up to 5 hours

post-exercise. These studies indicate the dramatic effect that muscle injury can have on muscle

force at low activation frequencies.

Highly trained endurance athletes display exceptional muscular endurance and typically

have greater percentages of slow-oxidative muscle fibers (56) when compared to SED. In

addition to less pronounced LFF, these slow-oxidative muscle fibers have been reported to be

less susceptible to contraction-induced muscle injury (22, 38). Also, it has been reported that

endurance training, specifically running, seems to provide protection from muscle injury due to

the eccentric component involved with this exercise modality (18, 57). If ET individuals, in fact

have greater percentages of slow-oxidative fibers, and are more protected from contraction–

induced muscle injury, then it is possible that they would be less prone to LFF following a bout

of exercise designed to injure skeletal muscle. However, a study by Skurvydas et al. (62) showed

no differences in LFF between long-distance runners, sprinters and untrained men after stretch-

shortening exercise, which consisted of 100 maximal drop jumps. They may have failed to show

differences between groups due to the fact that LFF was measured in the muscles of the thigh,

but other muscle groups were likely involved during exercise. Also, drop jumps performed may

not have induced substantial fatigue and/or muscle injury solely to the quadriceps muscle,

potentially not evoking enough LFF to distinguish differences between groups.

15

Surface neuromuscular electrical stimulation (NMES)

The use of surface neuromuscular electrical stimulation (NMES) is commonly used in

testing of able-bodied subjects as well as in those with neuromuscular disorders to facilitate

contraction of weak or paralyzed skeletal muscle. Activating skeletal muscle for the purpose of

functional movements and exercise through NMES is challenging as skeletal muscle fatigue can

occur rapidly. The profound fatigue that occurs with this modality, is likely due to altered motor

unit recruitment patterns (20, 35, 36), as well as synchronous activation of a given motor neuron

pool (1, 52). It is well established that the use of NMES requires greater energy demand and

consequently causes more fatigue when compared to similar voluntary efforts (28, 35, 69).

Utilizing high frequencies (40-100 Hz) of NMES leads to rapid onset of fatigue but force

typically returns to baseline values fairly quick during recovery. Alternatively, low frequency

NMES typically leads to less fatigue during contractions but recovery of force is prolonged (17,

31, 63).

It is believed that fast-twitch fibers are activated to a greater extent with NMES than

during voluntary activation at matched submaximal workloads (35, 69). The enhanced force loss

with NMES is thought to be due to the greater myofibrillar adensosine triphosphatase rates in

these fast-twitch muscle fibers as well as their rapid loss of phosphocreatine stores (65).

Research has focused on trying to limit skeletal muscle fatigue with the use of NMES systems in

able-bodied subjects as well as those with neuromuscular diseases. One such approach has been

experimentation with variable frequency trains (55, 64), which alter the pulse frequency within a

train of stimulation. Variable-frequency trains may show promise in future NMES systems,

although recent data shows that the efficacy of this stimulation in spinal cord injured (SCI)

patients appears to be limited (58). In summary, exercise evoked via NMES leads to greater

16

levels of fatigue, which contributes to low levels of work typically reported with traditional

NMES-evoked training, and generally translates into limited improvements in fitness and health.

Muscle fatigue and SCI

Muscle fatigue has been reported to be greater in individuals with SCI, as compared to

able-bodied subjects (5, 12, 30). The most widely supported mechanism explaining the high

levels of fatigue observed in SCI subjects has been attributed to the increased proportion of fast-

twitch fibers, which have greater ATP turnover rates compared to slow twitch fibers. “Slow to

fast” fiber conversion has been shown to occur after 1-2 years post-injury with increased

expression of myosin heavy chain IIa and IIx (8, 25), as well as faster contraction speeds (24,

54). Muscle biopsy data from m. tibialis anterior from SCI subjects 2-11 years post-injury

indicate significantly less slow-twitch fibers than controls (69% vs. 14%, respectively) and

smaller mean fiber cross-sectional area (40). It is reported that a near complete conversion from

‘slow to fast’ muscle occurs over a period of approximately 70 months post-injury (8). Along

with fiber type changes, studies have demonstrated decreases in mitochondrial size as well as

reduced oxidative enzyme activities (26, 40). Over time, the paralyzed skeletal muscle contains

smaller fibers that have a greater energy demand (greater contractile speed) and a lower capacity

to supply it, leading to greater muscle fatigue. These factors likely account for some of the

difference in fatigability between SCI and able-bodied, with other factors possibly including

altered calcium handling and/or muscle injury occurring during the onset of muscular

contractions.

Low frequency fatigue and SCI

Although many studies have examined fatigue during contractions in SCI, few have

examined force for several hours after a bout of NMES-evoked exercise. Since there is a

17

somewhat complete transformation from slow to fast contractile machinery with chronic SCI, as

well as an increased risk of contraction-induced muscle injury (5), it seems plausible that

paralyzed muscle would be more susceptible to LFF than able-bodied muscle. To our

knowledge, few studies have examined LFF in individuals with SCI. Shields et al. (61) examined

low frequency force loss and recovery in the soleus muscle of chronic (> 3 years post-injury) and

acute SCI (< 5 weeks post-injury). The fatiguing NMES protocol consisted of 330 ms trains

delivered every second for 3 minutes. During the fatigue protocol they measured twitch (1Hz)

and tetanus (20Hz) force every 30 seconds during fatiguing contractions and at 5 min post-

exercise. Immediately following exercise, peak twitch and tetanus torque was reduced ~80% and

75%, respectively in the chronic SCI compared to only ~ 14% and 16% in acute SCI group.

Twitch and tetanus force in acute SCI had fully recovered within 5 minutes whereas chronic SCI

force had only recovered to ~65% and 60% of pre-fatigue values at 1 and 20 Hz, respectively.

This study, however did not actually measure LFF because high frequency force was not

assessed. Also, the fatigue protocol did not evoke LFF in the acute SCI group because low

frequency force (1 and 20 Hz) had recovered fully within 5 minutes.

Another study by Shields’ group (60), which was nearly identical to the aforementioned

study, was performed on 8 individuals with SCI (7 chronic, 1 acute) but assessed force at several

frequencies before, immediately after, and at 5 and 15 min following a 4 min fatigue protocol.

The frequencies used to assess force before and after the fatigue protocol were 1, 5, 10, 15, 20,

30, and 40 Hz. Extracting data from their graphs and calculating the ratio of force produced at 10

and 40 Hz as an indicator of LFF, yields ratios of 0.49, 0.34, 0.24, and 0.22 for before,

immediately after, and 5 and 15 min following fatigue, respectively. Although this study

18

demonstrates LFF in individuals with SCI it is limited, as it did not measure LFF for substantial

periods after the fatigue protocol.

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23

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24

CHAPTER III

LOW FREQUENCY FATIGUE AFTER ELECTRICALLY EVOKED

CONTRACTIONS IN TRAINED AND UNTRAINED SUBJECTS

____________________ Mahoney ET, Dudley GA, and McCully K. To be submitted to European Journal of Applied Physiology.

25

Abstract

Low frequency fatigue (LFF) is defined as a relative loss of force at low activation

frequencies and is related to impairments in excitation-contraction coupling. The aim of this

study was to examine LFF in endurance trained (ET) and sedentary (SED) individuals. Ten ET

and nine SED subjects performed two separate neuromuscular electrical stimulation (NMES)

protocols to evoke fatigue of the quadriceps muscle of one thigh (experimental leg) with the un-

fatigued leg as a control. Protocol 1 (‘15 Min’) lasted 15 min and consisted of 3-sec isometric

contractions at 30 Hz with 6 sec between contractions. For protocol 2, stimulation was adjusted

so that fatigue in the SED group was matched to ET during the 15 min protocol (‘Low Matched’)

while fatigue in the ET group was matched to the SED group 15 min protocol (‘High Matched’).

Force was assessed at 20 Hz (P20) and 100 Hz (P100) and the ratio of P20/P100 was used to

evaluate LFF in both thighs before and up to 24 hours following fatigue. The SED group had

significantly greater reduction in force during the 15 Min fatigue protocol (p < 0.001). The SED

group had a greater magnitude of LFF with the 15 Min (p < 0.001) and High Matched (p <

0.020) conditions. The ET group did not recover faster than the SED group for any of the

conditions. Muscle pain 24 hours after the fatigue tests (~1.5 out of 10) may have influenced

LFF values. In summary, ET had less LFF than SED even when relative force loss was matched

between groups, suggesting that endurance trained muscle is more ‘protected’ from LFF.

Endurance trained muscle did not have faster recovery of LFF, but other factors such as muscle

injury make interpretations of the recovery difficult.

Key words: Low frequency fatigue, electrical stimulation, endurance training, calcium

impairment.

26

Introduction

Endurance trained (ET) athletes demonstrate superior fatigue resistance compared to

sedentary (SED) individuals. During exercise, ET subjects have less relative force loss over

contractions and recover faster from metabolic fatigue, which is due to enhanced oxidative

capacity (11, 17, 29). Extensive literature exists regarding muscular force decrements that occur

with metabolic fatigue between ET and SED. However, little is known about how endurance

training affects the prolonged recovery of force, which occurs independent of metabolic fatigue

and is most evident at low frequencies of electrical stimulation (8, 22). This phenomenon is well

documented and is commonly known as low frequency fatigue (LFF), which has been shown to

last 6-8 hours or longer (8, 18, 25).

Low frequency fatigue is defined as a preferential loss of force at low stimulation

frequencies (ie. 20 Hz) compared to high frequencies (ie. 100 Hz). It is commonly assessed by

the ratio of force produced at low and high activation frequencies following muscular fatigue

(25, 27, 28). Although the exact mechanism is unclear, LFF is thought to be due impaired

excitation-contraction coupling with evidence specifically pointing to reduced calcium release

from the sarcoplasmic reticulum at low stimulation frequencies following fatigue (15, 30, 31). As

stated, LFF can exist for hours after fatiguing exercise, but many of the previous studies have not

followed it throughout its full recovery.

Few studies have examined how endurance training might affect LFF after exercise. To

our knowledge, only one study has directly examined LFF between ET and sedentary

individuals. A study by Skurvydas et al. (24) showed that LFF was not different between long

distance runners, sprinters, and untrained men. In this study, LFF was assessed in the thigh but

additional muscle groups were likely recruited during jumping exercise, which may limit these

27

findings. Also, the exercise may not have induced substantial fatigue/injury solely to the

quadriceps muscle, potentially not evoking enough LFF to distinguish differences between

groups.

Low frequency fatigue has been shown to be influenced by metabolic changes within

muscle (16, 19). Pronounced fatigue causes large increases in inorganic phosphate and hydrogen

ions. It is hypothesized that high levels of inorganic phosphate may be taken up into the

sarcoplasmic reticulum, where it may precipitate with calcium (10). The formation of calcium

phosphate would lower free calcium concentrations in the sarcoplasmic reticulum, thus reducing

calcium release. A study by McCully et al. (16) determined that protocols that elicit high levels

of fatigue, and therefore metabolic byproducts, can increase the magnitude of LFF observed

during recovery. With regard to ET and SED individuals, previous literature would indicate that

when these two groups perform identical protocols that SED would incur greater metabolic

fatigue and force loss. For these reasons, it seems plausible that SED individuals would have

greater LFF following contractions, compared to ET subjects.

The aim of the present study was to examine how endurance training influences both the

magnitude and recovery of LFF. Endurance trained and SED subjects performed fatigue tests

that were either identical in the number of contractions or in percentage force loss. We

hypothesize that ET subjects would have: 1) less magnitude of LFF and 2) faster recovery of

LFF compared to SED subjects following the fatigue tests.

Methods

Subjects:

Ten ET (two females) and nine SED (four females) subjects participated in this study.

The participants had no recent history of lower extremity pathology and gave written consent

28

prior to testing. This study was approved by the Institutional Review Board at the University of

Georgia. Sedentary subjects were performing one or fewer days of light aerobic exercise per

week. All of the ET subjects were performing vigorous aerobic exercise (~60-90% of heart rate

maximum) at least 5 hours per week. These subjects consisted of runners, cyclists and/or

triathletes. The recruitment of the ET group was based on the amount of endurance exercise the

subject participated in, rather than absolute aerobic fitness (ie. VO2 max). However, six of the ten

ET subjects had recently achieved VO2 max values ranging from 57 to 69 ml/kg/min during a peak

cycle ergometer test (personal communication Dr. Jonathan Wingo). All subjects were asked to

refrain from intense or unaccustomed exercise 24 hours prior to testing and to remain relatively

inactive for the total duration of testing. In addition, subjects were asked to abstain from caffeine

on testing days as it has been shown to affect muscular force at low activation frequencies (28).

Force measurements:

For isometric contractions of the thigh, subjects were seated in a custom-built force chair

with the hip and knee secured at approximately 70° of flexion. Both legs were firmly secured to a

rigid lever arm with an inelastic strap to ensure that the knee extensors could only perform

isometric contractions. The moment arm was established by placing a load cell (model 2000A;

Rice Lake Weighing Systems, West Coleman Street, Rice Lake, Wisconsin, USA) parallel to the

line of pull and perpendicular to the lever arm. Force was recorded from the load cell using a

MacLab A-D converter (model ML 400; ADInstruments, Milford, Mass., USA) sampling at

100 Hz and interfaced with a portable Macintosh computer (Apple Computer, Cupertino, Calif.,

USA). All force tracings were displayed and recorded on a computer.

29

Electrical stimulation (NMES):

A high voltage electrical stimulation unit (Rich-Mar Theratouch 4.7) was used to assess

LFF and to evoke contractions during fatigue protocols. Stimulation was delivered through two

electrodes (8×10-cm) to evoke isometric contractions of the quadriceps femoris muscle group.

One electrode was placed 2-3 cm above the superior aspect of the patella over the vastus

medialis muscle. The second electrode was placed lateral to and 30 cm above the patella over the

vastus lateralis muscle. Permanent ink marker was used to trace the electrodes to ensure the same

placement for the 24-hour assessment of LFF, or at any other time the electrodes were taken off

during testing.

Force of the quadriceps femoris muscle group, elicited at 20 Hz (P20) and 100 Hz (P100)

was measured before, immediately after, and at 10, 20, and 60 min, as well as 2, 4, 6 and 24

hours following a fatigue protocol. At each time point, the quadriceps femoris muscle group was

given two 1-sec contractions at 20Hz followed immediately by two 1-sec contractions at 100 Hz.

These contractions were elicited to potentiate the muscle. Approximately 10 sec following these

four contractions, a 1-sec contraction at 20 Hz was given followed immediately by a 1-sec

contraction at 100 Hz for the assessment of LFF and these will be referred to as ‘evaluation

stimulations’. These 1-sec contractions at 20 and 100 Hz were elicited three times for each

assessment. The ratio of P20/P100 was calculated and the percentage reduction in this ratio was

used to evaluate LFF (8, 32). A greater percent reduction in P20/P100 from baseline values (pre-

fatigue) indicates greater LFF.

Fatigue tests for both ET and SED subjects were elicited via NMES. All contractions

during the fatigue tests were evoked with 30-Hz trains of 450-µs biphasic pulses. The fatigue

tests were performed on one thigh (experimental leg). The contralateral, un-fatigued leg served

30

as control. The experimental leg was assigned by counterbalancing dominant/non-dominant leg

for each subject and was kept the same for each subject for both test sessions. The measurements

of P20 and P100 were assessed in both thighs and P20/P100 values from the control leg were

used statistically to account for the effects of using NMES to evaluate LFF.

Experimental protocol:

Familiarization. Subjects were seated in the custom designed isometric force chair and

their legs and torso were firmly strapped into the chair. Subject’s legs were cleaned with alcohol

and two electrodes were placed on each thigh as previously described. Electrical stimulation at a

frequency of 100 Hz was given to evoke 1-3 sec isometric contractions of the knee extensors at

increasing current levels until ~22.7 kg of isometric force was produced. If this level of NMES

current was tolerated well and subjects were still interested in participation, they were asked to

return to the laboratory 1-2 days later for their first test session.

Test Session 1. Subjects were seated in the custom designed isometric force chair and


and two electrodes were placed on each thigh as previously described. Electrical stimulation

amplitude at 100 Hz was slowly increased over several 1-sec contractions to achieve ~ 22.7 kg of

isometric force in each thigh. After the NMES amplitude was determined, it remained the same

in each leg throughout the duration of each test session for the measurements of P20 and P100,

as well as for fatigue tests. As stated previously, P20 and P100 were measured before,

immediately after, and at 10, 20, and 60 min, as well as 2, 4, 6 and 24 hours following an NMES-

evoked fatigue test.

For test session 1, the NMES-evoked fatigue test for both ET and SED lasted 15 min and

consisted of 3-sec isometric contractions of the thigh with 6-sec rest between contractions. This

31

allowed for a total of one hundred 3-sec contractions, which were evoked at the same NMES

amplitude used for measurement of P20 and P100. This was designated the ’15 Min’ condition.

Test Session 2. Subjects returned for the second test session within a minimum of two

weeks following the first test session. The procedures and evaluation were the same as test

session 1 except that a different fatigue protocol was used. ET subjects performed a protocol

designed to produce the same amount of fatigue as the average fatigue value for the SED

subjects during the 15 min protocol. To achieve this, the ET group performed a fatigue protocol

with a duty cycle of 60% (3 sec on/2 sec off) lasting 8.3 min, allowing ET subjects to perform

the same number of contractions as in test session 1. This was designated the ‘High Matched’

condition. The SED subjects performed a protocol designed to produce the same amount of

fatigue as the average fatigue value for the ET group during the 15 min protocol. The duty cycle

for this fatigue test was the same as session 1, (33%; 3 sec on/6 sec off) and lasted 3-8 min,

which was dependent on individual force reductions in the SED group. This protocol was

designated the ‘Low Matched’ condition.

Assessment of muscle pain:

Muscle pain was evaluated in both the experimental and control leg based on a 10-point

Likert-type scale. Subjects were asked to rate the intensity of leg muscle pain from 0 “no pain at

all” to 10 “worst possible pain imaginable”. Subjects verbally reported these pain ratings before,

and at 24 and 48 hours following the NMES-evoked fatigue test.

Analysis of force tracings:

Force tracings for the first and last three contractions of the fatigue protocols were

analyzed by measuring force-time integrals (FTI). Fatigue was calculated as follows:

32

Fatigue = [FTI (1st 3 contractions) – FTI (last 3 contractions)] ÷ FTI (1st 3 contractions)

Single contractions for the assessment of P20 and P100 were always 1 sec in duration. For each

force tracing at 20 and 100 Hz, the average value for the first 0.5 sec after the initial rise was

analyzed. The highest mean force at 20 Hz and 100 Hz was used in the calculation of the

P20/P100 ratio at each time point.

Statistical Analyses:

Data analyses were performed using SPSS version 13.0 (SPSS Inc., Chicago, IL).

Descriptive statistics are presented in text, figures and tables as mean ± SD unless otherwise

noted. Independent t-tests were used to assess potential differences between the groups in subject

age, body mass, and percent force loss during NMES-evoked fatigue protocols. In the t-test

analyses, the homogeneity of variance was tested using Levene’s test for equality of variance.

Simple linear regression was used to examine relationships between 24 hour LFF (as percent

reduction in P20/P100 ratio) and muscle pain at 24 hours. Statistics were adjusted accordingly if

the assumption of equality of variance was violated.

For all analyses, the ratio of the P20/P100 was calculated for each time point. These time

points were then reported, as the percentage reduction from baseline and from here on will

represent LFF. The time points collected in the first 60 minutes post-fatigue were used to assess

the magnitude of LFF. These time points were selected because theory and prior empirical

research suggest that LFF can last 1 hour or longer when fatiguing contractions are performed

without causing muscle injury (5, 8). Also, 1 hour was selected as this tests our first hypothesis

about the magnitude of LFF after a fatigue protocol.

33

A separate analysis for magnitude of LFF was performed for each of the testing

conditions (ie. 15 Min, High Matched and Low Matched protocols). In each analysis, magnitude

of LFF in the experimental leg was analyzed using a 2 (group: SED, ET) x 4 (time: 0, 10, 20, and

60 minutes post-fatigue) mixed model ANCOVA with a repeated measure on the time variable.

P20/P100 ratio values for the control leg were used as varying covariates in an attempt to control

for the effects of using NMES to assess LFF.

A separate analysis for recovery of LFF was performed for each of the testing conditions

(ie. 15 Min, High Matched and Low Matched protocols). Recovery of LFF was analyzed using a

2 (group) x 8 (time: 0, 10, 20, 60 minutes; 2, 4, 6, and 24 hours) trend analysis in order to

examine differences in the rate of recovery between groups after the fatigue protocol. The

control leg LFF values were used as covariates in the trend analyses.

Perception of thigh muscle pain 24 and 48 hours after the fatigue protocol was used as an

indirect measure of muscle injury. A separate analysis for thigh muscle pain intensity was

performed for each of the experimental conditions (ie. 15 Min, High Matched and Low Matched

protocols). Pain data were analyzed using a 2 (group) x 3 (time: Pre, 24 & 48 hours post-fatigue)

ANOVA with repeated measure on the time variable.

Simple t-tests were used to assess potential differences in 100 Hz force between baseline

and 24-hour post-fatigue data within groups for each condition. Significant reductions in 100 Hz

muscle force at 24 hours were also used as an indicator of muscle injury.

All mixed model analyses were based on the F-statistic. Effects sizes for F-statistics were

expressed as partial eta-squared (η2). The Greenhouse-Geisser epsilon (ε) was reported and

degrees of freedom were adjusted when the sphericity assumption was violated (ie. if Mauchly’s

34

test of sphericity was statistically significant at p < 0.05). The familywise error rate was

controlled using the Bonferroni adjustment when tests of simple effects were conducted.

Results

Participant Characteristics:

Subject demographics are shown in table 3.1. No statistically significant differences

existed between groups for age (F (17) = 0.142, p = 0.244) or body mass (F (17) = 5.28, p =

0.656). Ten ET subjects completed test session 1 (‘15 Min’ condition). Nine of these ET

subjects returned to complete test session 2 (‘High Matched’ condition). The ET subject that did

not finish the study had scheduling difficulties and was unable to return for test session 2. Nine

SED subjects completed both test session 1 and 2 (‘15 Min’ and ‘Low Matched’ conditions). No

adverse events occurred during this study.

Force Reduction During NMES-Fatigue Protocols:

There was a significantly greater percent reduction in force in the SED group than the ET

group with the 15 Min fatigue protocol (-49.6 ± 9.5% vs. -27.6 ± 9.4%, respectively) (p < 0.001).

There was not a statistically significant difference in force loss between the SED and ET groups

in the High Matched (p = 0.57) and Low Matched (p = 0.99) conditions. Percent reduction in

force-time integrals during these NMES-evoked fatigue protocols for the ET and SED groups are

shown in Figure 3.1.

The duration of the ET High Matched fatigue protocol was 8.3 minutes, and used a shorter

duty cycle (3 s on/ 2 s off) in order to produce the same amount of fatigue as the average fatigue

value for the SED group during the 15 min protocol (~50%). The SED Low Matched fatigue test was

performed at the same duty cycle as the 15 min protocol (3 s on/ 6 s off) and lasted 4.8 ± 1.5

35

min, which was dependent on individual force reductions in this group. This protocol was used

to produce the same amount of fatigue as the ET group during the 15 min protocol (~28%).

Force Tracings:

Force tracings at 20 Hz and 100 Hz are shown in Fig. 3.2 for one sedentary subject before

and 1-hour following a fatigue test. Using the ratio of these forces allowed for quantification of

LFF. One hour after the fatigue protocol, 100 Hz force has returned to near baseline values,

whereas 20 Hz force is still depressed substantially.

100 Hz Force:

Percent change 100 Hz force values after a fatigue protocol are shown in Figure 3.3 (a-c) for 15

Min (a) and High Matched (b) conditions between ET and SED over 24 hours post-fatigue.

These data have been statistically adjusted for the control leg but no other statistical tests were

performed for these data. Return of 100 Hz force to baseline values shortly after exercise can be

used to assess recovery of metabolic fatigue.

LFF in the Control Leg:

Figure 3.4 shows the percent change from baseline P20/P100 values in the control leg for

the 15 Min condition in ET and SED groups. Both groups showed small amounts of LFF (8-

12%) in the control leg that remained constant over the time of the experiment. Data in the

control leg for the High and Low Matched conditions yielded similar responses (data not shown).

No statistical tests were performed for LFF in the control leg. In this study, LFF data from the

‘control leg’ will be used to statistically adjust for LFF values in the ‘experimental leg’. This

allowed us to determine what affect the fatigue protocol has on the response independent of the

evaluation stimulations.

36

Magnitude of LFF:

The magnitude of LFF, expressed as percent change from baseline P20/P100 ratio values

across the first 60 minutes post-fatigue, was examined for the 15 Min, High Matched and Low

Matched conditions (Figure 3.5a-c). No significant group x time interaction was found for the 15

Min (F (3, 50) = 0.43, p = 0.730, η2 = 0.025), High Matched (F (3, 47) = 0.96, p = 0.420, η2

=

0.058) or Low Matched (F (3, 50) = 1.71, p = 0.177, η2 = 0.093) conditions.

15 Min Condition. The magnitude of LFF was significantly greater in the SED group

compared to ET, based on a main effect for group (F (1,16) = 28.50, p < 0.001, η2 = 0.640), but

not time effect (F (3,50) = 1.44, p = 0.243, η2 = 0.079) for the 15 Min condition (Fig. 3.5a).

High Matched Condition. The magnitude of LFF was significantly greater in the SED

group compared to ET for the High Matched condition (Fig. 3.5b). This was based on a

significant main effect for group (F (1,15) = 6.74, p = 0.020, η2 = 0.310). In addition, there was a

significant main effect for time (F (3, 47) = 3.34, p = 0.027, η2 = 0.176) for this condition.

Low Matched Condition. The magnitude of LFF was not different between SED and ET

for the Low Matched condition. This was based on a lack of a significant main effect for group

(F (1, 16) = 0.20, p = 0.660, η2 = 0.012) (Fig. 3.5c). In addition, there was no significant main

effect for time (F (3,50) = 1.57, p = 0.208, η2 = 0.086) for this condition.

Recovery of LFF:

Trend analysis was used to examine difference between groups in the recovery of LFF

across the 24-hour period following the fatigue protocols (ie. 15 Min, High Matched and Low

Matched).

15 Min Condition. The recovery of LFF was significantly faster in SED compared to ET.

This was based on a significant group by trend interaction for the linear recovery of LFF over the

37

24-hour period between groups following the 15-minute fatigue protocol (F (7,135) = 2.77, p =

0.010, η2 = 0.126), which is shown in Fig. 3.6a. Decomposition of the interaction showed that

SED group recovered from LFF at a 9% greater rate than the ET group (t = 4.22, p < .001),

which was based on the differences in slopes for the linear trend between groups. Examination of

the data shows that the ET had only slight recovery of LFF over the 24 hours.

High Matched Condition. The recovery of LFF was not different between SED and ET

groups for the High Matched condition (Figure 3.6b). This was based on a lack of significant

group by trend interaction over the 24-hour post-fatigue period (F (7,127) = 0.72, p = 0.652, η2 =

0.038). However, there was a significant main effect for group (F (1, 127) = 18.73, p < 0.001, η2

= 0.129) and time (F (7, 127) = 13.03, p < 0.001, η2 = 0.418) for the recovery of LFF in the High

Matched condition.

Low Matched Condition. The recovery of LFF was not different between SED and ET

groups for the Low Matched condition (Figure 3.6c). This was based on a lack of significant

group by trend interaction over the 24-hour post-fatigue period (F (7,135) = 1.57, p = 0.149, η2 =

0.075). There was a significant main effect for time (F (7,135) = 5.50, p < 0.001, η2 = 0.222) but

not for group (F (1,135) = 1.62, p = 0.205, η2 = 0.012) for the recovery of LFF in the Low

Matched condition.

Indicators of Muscle Injury: Muscle pain ratings in the experimental leg in ET and SED groups at 24 and 48 hours

post-fatigue are shown in Table 3.2. (Pain was ‘0’ in all subjects before testing.) No pain was

reported for the control leg at pre, or at 24 or 48 hours post-fatigue. There were no significant

differences in pain ratings between groups at any time point. No significant group x time

interaction for pain was found for the 15 Min (F (2, 34) = 1.04, p = 0.364), High Matched (F (2,

38

32) = 0.785, p = 0.465) or Low Matched (F (2, 34) = 0.785, p = 0.460) conditions. Similarly, no

significant main effect for group was found for any of the three conditions (F (1, 17) = 1.65, p =

0.216; F (1, 16) = 1.129, p = 0.304; F (1, 17) = 0.831, p = 0.375 for 15 Min, High and Low

Matched, respectively.) However, all conditions demonstrated a significant main effect for time

(F (2, 34) = 37.39, p < 0.001), (F (2, 32) = 26.95, p < 0.001) and (F (2, 34) = 26.38, p < 0.001)

for 15 Min, High Matched, and Low Matched conditions, respectively).

Significant reductions in 100 Hz muscle force at 24 hours (from baseline values) were

used as an indicator of muscle injury. There were no significant within-group reductions in 100

Hz force at 24 hours compared to baseline values for any condition, indicating that muscle injury

was likely minimal. (p values = 0.092, 0.354, 0.397, and 0.230 for ET 15 Min, SED 15 Min, ET High

Matched, and SED Low Matched protocols, respectively).

Association between LFF and muscle pain:

LFF values and pain ratings at 24 hours post-fatigue in the experimental leg were used to

assess possible associations between LFF and muscle pain in the current study. There was a

significant linear relationship between 24-hour measures of LFF (as percent reduction in

P20/P100 ratio) and 24 h pain ratings for the 15 Min condition (r = -0.494, p = 0.032). This

indicates that approximately 24% of the variance in LFF at 24 h can be explained by muscle pain

or more indirectly, muscle injury. No significant relationships were found for these same

variables for the High Matched (r = -0.313, p = 0.207), and Low Matched (r = -0.064, p = 0.795)

conditions.

Discussion

The main finding of this study was that the SED group had a greater magnitude of LFF

compared to the ET group after fatigue protocols matched either to the total number of

39

contractions (15 Min) or the total amount of fatigue (High Matched). This is an important finding

as no differences in LFF have previously been reported between SED and ET subjects. In

addition, our results are consistent with the numerous studies that have shown that with the same

NMES-evoked or voluntary protocol that the total amount of fatigue is less in ET compared to

SED individuals (17, 29). In fact, our SED group had nearly double the amount of force loss

compared to ET (-49.6 ± 9.5% vs. -27.6 ± 9.4%, respectively) after the 15-minute fatigue

protocol. Regardless of whether fatigue was matched between groups, ET subjects appear to be

less susceptible to LFF following NMES-evoked contractions.

There are only two previous studies that have examined how aerobic fitness affects LFF

(3, 24) and the results are difficult to compare to our study. Skurvydas et al. (24) examined LFF

in long-distance runners, sprinters and untrained men after voluntary, jumping exercise. They

demonstrated substantial LFF immediately after, and 20 minutes following exercise in all groups,

but no between-group differences were observed. In this study, LFF was assessed in the thigh but

additional muscle groups were likely recruited during exercise, which may limit their findings.

Babcock et al. (24) examined LFF of the diaphragm muscle in highly fit and fit subjects before

and after an intense bout of running. They showed that both groups had significant LFF at 0 and

20 minutes post-fatigue, but no differences existed between groups at any time point. Both of

these two studies used voluntary, whole body exercise and then assessed LFF in one muscle or

muscle group, whereas the current study used NMES in the thigh muscles for exercise and for

assessment of LFF. Mixed findings between the previous studies and ours may be related to the

use of NMES-evoked exercise, which causes profound fatigue compared to voluntary exercise

and is likely due to altered motor unit recruitment patterns (9, 13, 14), as well as synchronous

activation of a motor units (1, 21). Also, it has been reported that NMES-evoked contractions

40

cause greater LFF than do voluntary ones (22), which may be responsible for the large

magnitude of LFF, and the differences observed between ET and SED in the present study.

A factor that may explain differences in the magnitude of LFF observed is the amount of

metabolic stress that occurred during the fatigue protocol (16). A study by McCully et al. (16)

used an in vivo rat model to examine the potential role that metabolic byproducts might have on

LFF. They examined muscular force and metabolic byproducts during and following bouts of

electrical stimulation. Metabolic fatigue was based on concentrations of inorganic phosphate and

LFF was assessed by force produced at low (20Hz) and high frequency (120Hz) electrical

stimulation. They determined that protocols that elicit high levels of fatigue, and therefore

metabolic byproducts, can increase the magnitude of LFF observed during recovery. This study

supports our results in that higher levels of fatigue, as was the case for SED after the 15 min

protocol, lead to greater initial amounts of LFF.

Although not quantitatively measured in this study, SED subjects likely incurred greater

metabolic stress during the 15 min fatigue protocol as force loss was nearly double that of ET.

Even when force loss was the same between groups, ET likely have less disruption in overall

metabolism due to enhanced mitochondrial function (17), which would blunt the production of

inorganic phosphate and hydrogen ions. It has been suggested that at high concentrations,

inorganic phosphate can enter the sarcoplasmic reticulum and precipitate with calcium (10)

therefore reducing available calcium for release. With a similar metabolic environment, muscle

force would be most affected at low activation frequencies and may be a plausible explanation as

to why the magnitude of LFF was more pronounced in SED subjects.

Recovery of LFF over 24 hours was not greater in ET for any of the conditions. However,

trend analysis revealed that the SED group actually recovered faster (~9%) from LFF compared

41

to ET following the 15-minute fatigue protocol. In fact, the ET had minimal recovery of LFF

over 24 hours after this fatigue protocol. This was unexpected, as we predicted that recovery

would be faster in ET subjects. Although overwhelming evidence shows that ET subjects recover

faster from metabolic fatigue, it is apparent that the recovery of LFF is distinctly different. It

seems plausible that SED recovered faster only because of their large magnitude of LFF.

Despite this finding, ET subjects are still better at maintaining low frequency force after fatigue,

which is important as most submaximal, voluntary contractions are elicited at low frequencies

(15-20 Hz)(20).

To our knowledge, no studies have assessed LFF at several time points throughout a

prolonged recovery period (ie. 24 hours) between ET and SED subjects, as was done in the

current study. Regardless of the groups examined, few studies have followed the recovery of

LFF more than an hour after exercise, even though it is possible to last several hours or even

days (5, 8, 12, 18). Skurvydas et al. (26) reported that after a 60 sec isometric sustained maximal

contraction of the quadriceps muscle, significant LFF was observed at 15 minutes post-exercise

in healthy untrained men. In a study by Blangsted et al. (5), healthy subjects performed static

wrist extension at 10% of maximal voluntary contraction for 10 minutes. They demonstrated that

their protocol caused significant LFF that lasted up to their final assessment at 2.5 hours post-

exercise. Besides fatigue, other factors such as muscle injury have been shown to affect LFF (7,

12, 18, 23), which seems to have a more profound, long-lasting effect on the response. Since the

origin of LFF has been attributed to disruption in the excitation-contraction coupling process,

studies have demonstrated that muscle injury can impair calcium handling and affect muscular

force, most predominately at low frequencies (2, 6, 18). Child et al. (7) examined the recovery of

LFF after a bout of eccentric contractions of the thigh, which were evoked by NMES and caused

42

significant muscle pain and injury. They showed that subjects had significant LFF up to 3 days

following this type of damaging exercise. It is apparent from such studies that decrements in low

frequency force can be severe with muscle injury and can last for days.

Prior to the start of this study, we believed that using NMES to evoke isometric

contractions would not cause muscle injury in able-bodied subjects based on previous research

(4). However, we believe that small amounts of muscle injury may have occurred in this study

and confounded our results, making interpretations of the recovery of LFF difficult. In the

current study, nearly three-fourths of all subjects tested reported low levels of muscle pain 24

hour post-fatigue, which would indicate that muscle injury, albeit minor, might have occurred.

Although 100 Hz force was not significantly reduced at 24 hours, P20/P100 ratios at 24 hours

were still reduced 10-15% from baseline values in all groups and conditions, which can be best

explained by the long-lasting effects of muscle injury. It is likely that some combination of

metabolic fatigue, as well as muscle injury played a role in the LFF response. However, the

current study does not allow us to determine their relative importance.

Several potential limitations exist for this study. First, it was not feasible to have subjects

remain in the force chair for 24 hours and therefore they had to re-enter the force chair several

times throughout the study, which may have increased variability of our force measures. To

counter this we tried to be as meticulous as possible by setting up subjects in the force chair the

same way each time they arrived and by using the non-fatigued leg as control. Secondly, surface

NMES was used and changes in the properties of the electrodes and/or the skin over the course

of the study may have altered conductance of electrical current and generation of muscular force.

Subjects were asked to remain relatively inactive over the course of the day and the electrodes

were traced to ensure the same placement for the 24-hour assessment of LFF, or at any other

43

time the electrodes were taken off during testing. Third, this study used submaximal electrical

current to evoke muscular contractions of the quadriceps muscle and therefore only small

portions of this muscle group were likely recruited. Submaximal current was used to limit the

pain associated with its use in individuals with normal sensory function. Future studies may want

to stimulate smaller muscles to maximize the chance of activating the entire muscle. Regardless,

this study shows that there are distinct differences in the magnitude of LFF between ET and SED

subjects.

Conclusion

In summary, ET had less magnitude of LFF compared to SED even when total fatigue

was matched, suggesting that endurance trained muscle is more ‘protected’ from LFF. Endurance

trained muscle did not have faster recovery of LFF, but other factors such as muscle injury make

interpretations of the recovery difficult. Future studies are needed to utilize voluntary

contractions to examine potential differences in LFF between ET and SED individuals. The

performance of voluntary contractions will reduce the chance that muscle injury will occur and

any differences in LFF can be attributed to the metabolic perturbations between these two

groups.

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47

Figure 3.1. Percent reduction in force-time integrals during fatigue protocols in endurance

trained and sedentary subjects. Values are Mean ± SD; * p ≤ 0.05 for SED vs. ET.

49

Figure 3.2. Representative force tracings at 20 Hz and 100 Hz for one sedentary participant pre-

and 1-hour post-fatigue.

50

Figure 3.3 (a-c). 100 Hz force values (% initial) for 15 Min (a), High Matched (b) and Low

Matched (c) conditions in ET and SED. Return of 100 Hz force to baseline values shortly after

exercise can be used to assess recovery of metabolic fatigue. Values are Mean ± SD and have

been statistically adjusted for the control leg. (ET = open circles; SED = filled triangles). No

statistical tests were performed for these data.

51

Figure 3.4. Control leg LFF values over 24 hours post-fatigue in ET and SED for the 15 Min

condition (High and Low Matched not shown). Values are Mean ± SD. (ET = open circles; SED

= filled triangles). No statistical tests were performed for these data.

52

Figure 3.5 (a-c). Magnitude of LFF over 1-hour post-fatigue for 15 Min (a), High Matched (b)

and Low Matched (c) conditions in ET and SED. Values are Mean ± SD and have been

statistically adjusted for the control leg. * p ≤ 0.05, Main effect for group (ET = open circles;

SED = filled triangles).

53

Figure 3.6 (a-c). Recovery of LFF for 15 Min (a), High Matched (b) and Low Matched (c)

conditions in ET and SED over 24 hours post-fatigue. Values are Mean ± SD and have been

statistically adjusted for the control leg. * p ≤ 0.05, group by trend interaction with SED

recovering from LFF at a greater rate. (ET = open circles; SED = filled triangles).

54

CHAPTER IV

LOW FREQUENCY FATIGUE IN INDIVIDUALS WITH SPINAL CORD INJURY

____________________ Mahoney ET, Dudley GA, and McCully K. To be submitted to Muscle and Nerve

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Abstract

Low frequency fatigue (LFF) is defined as a prolonged, preferential loss of muscular

force at low activation frequencies and is related to impairments in excitation-contraction

coupling. The primary aim of this study was to examine the magnitude and recovery of LFF

following a fatigue test in spinal cord injured (SCI) and sedentary able-bodied subjects. Nine

able-bodied and nine SCI subjects performed a single neuromuscular electrical stimulation

(NMES) protocol to evoke fatigue of the quadriceps muscle of one thigh (experimental leg) with

the un-fatigued leg as a control. The fatigue test for able-bodied subjects lasted 15 min and the

duty cycle was 33% (3 s On/6 sec Off). For SCI, stimulation was adjusted so that fatigue was

matched to the able-bodied group during the 15 min protocol. Force was assessed at 20 Hz (P20)

and 100 Hz (P100) and the ratio of P20/P100 was used to evaluate LFF in both thighs before and

up to 24 hours following fatigue. Results indicated that force loss during the fatigue protocol was

not different between groups. In the experimental leg, the magnitude of LFF was not

significantly different between groups. Recovery of LFF over the 24 hours was significantly

greater in able-bodied compared to SCI in both the experimental (p < 0.001) and control leg (p <

0.001). When LFF values in the experimental leg were corrected for LFF values in the control

leg, no differences existed between groups (p=0.064). The able-bodied group showed a gradual

recovery of LFF in the experimental leg, while the SCI did not. These results demonstrate that

SCI are more susceptible to LFF than able-bodied subjects. Importantly, just testing for LFF

produced substantial LFF in the SCI group, and can account for a substantial portion of the

response. We propose that muscle injury is the main factor responsible for the incomplete

recovery of LFF in the SCI group and future studies need to test whether fatigue in SCI subjects

actually represents the accumulation of muscle injury rather than fatigue.

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Key words: Low frequency fatigue, spinal cord injury, electrical stimulation, calcium

impairment, muscle injury.

Introduction

Spinal cord injury (SCI) leads to dramatic alterations in skeletal muscle morphology and

function depending on the level of injury. The use of neuromuscular electrical stimulation

(NMES) is commonly used in those with SCI to facilitate contraction of the paralyzed

musculature for both rehabilitation as well as fitness conditioning. It is well established that the

use of NMES requires greater energy demand and consequently leads to a disproportionate

amount of fatigue when compared to similar voluntary efforts (1, 11, 16, 36). In addition, muscle

fatigue has been reported to be greater in individuals with SCI during NMES-evoked

contractions, as compared to able-bodied controls (2, 5, 13, 23). The affected musculature of SCI

can undergo dramatic losses in force during NMES-evoked contractions, equating 2-4 fold

greater relative force loss than able-bodied (5, 13).

Although a great deal of literature exists regarding fatigue during NMES-evoked

contractions in SCI and able-bodied subjects, few studies have examined recovery of muscular

force after a fatiguing bout of exercise. After exercise, prolonged decrements in muscular force

have been documented, and occur mainly at low frequencies of electrical stimulation (7, 26).

This has appropriately been termed low frequency fatigue (LFF) and is defined as a preferential

loss of force at low activation frequencies (ie. 20 Hz) compared to high frequencies (ie. 100 Hz).

Low frequency fatigue is commonly assessed by the ratio of force produced at low and high

activation frequencies following muscular fatigue (7, 28, 32, 34). Although the exact mechanism

is unclear, LFF is thought to be due impaired excitation-contraction coupling with evidence

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specifically pointing to reduced calcium release from the sarcoplasmic reticulum at low

stimulation frequencies following fatigue (7, 8, 20, 38).

Following muscular contractions, LFF is evident in both fast and slow twitch muscle

fibers of animals, as well as humans (14). However, it has been reported that fast-glycolytic

fibers demonstrate greater LFF than do fast-oxidative fibers (25, 27). Powers & Binder (1991)

reported that fatigue resistant motor units from cat flexor digitorum muscles exhibited less

pronounced LFF than fast intermediate and fast-fatigable motor units after electrically stimulated

contractions. “Slow to fast” fiber conversion has been shown to occur 1-2 years after SCI with

increased expression of myosin heavy chain IIa and IIx (3, 10), as well as faster contraction

speeds (9, 29). This shift to predominantly fast-twitch muscle with SCI may predispose them to

higher levels of LFF following contractions.

Low frequency fatigue has been shown to be influenced by metabolic changes within

muscle (19). Pronounced fatigue causes large increases in inorganic phosphate and hydrogen

ions. It is hypothesized that high levels of inorganic phosphate may be taken up into the

sarcoplasmic reticulum, where it may precipitate with calcium (8). The formation of calcium

phosphate would lower free calcium concentrations in the sarcoplasmic reticulum, thus reducing

calcium release. A study by McCully et al. (19) determined that protocols that elicit high levels

of fatigue, and therefore metabolic byproducts, can increase the magnitude of LFF observed

during recovery.

Low frequency fatigue is more pronounced when muscle injury has occurred (6, 15, 22,

27). Since the origin of LFF has been attributed to disruption in the excitation-contraction

coupling process, studies have demonstrated that muscle injury can impair calcium

release/reuptake rates and affect muscular force (4), most predominately at low activation

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frequencies (22). Unloaded skeletal muscle, as in SCI, has been shown to be more susceptible to

muscle injury upon reloading (2, 17, 21, 24, 33, 37). A study by Bickel et al. (2) showed that

individuals with long-term SCI had significant muscle injury in the quadriceps muscle following

NMES-evoked isometric contractions, whereas able-bodied had no injury. Since SCI have

greater metabolic impairment, increased risk of contraction-induced muscle injury, as well as a

near complete transformation from slow to fast twitch muscle fibers, it seems plausible that they

would have greater LFF following contractions, compared to ambulatory individuals.

To our knowledge, no studies have examined LFF between able-bodied subjects and

those with SCI. We have recently shown that sedentary, able-bodied subjects had a greater

magnitude of LFF compared to endurance-trained athletes after equivalent levels of fatigue.

(Mahoney et al., in preparation). The aim of the present study is to examine the magnitude and

recovery of LFF between SCI and able-bodied subjects following a fatigue protocol designed to

match force loss. We hypothesize that able-bodied subjects will have: 1) less magnitude of LFF

and 2) a faster recovery of LFF compared to SCI subjects.

Methods Subjects:

Nine SCI (2 females) and nine able-bodied (four females) subjects participated in this

study. Motor and sensory function of SCI participants was previously assessed by the American

Spinal Association (ASIA) classification system. All SCI participants were non-ambulatory and

consisted of those with complete and incomplete spinal lesions ranging from C5 to T9. Mean

duration of spinal injury was 13.6 ± 12.2 years. The participants gave written consent prior to

testing. This study was approved by the Institutional Review Boards of the University of Georgia

and Shepherd Center. Able-bodied subjects were performing one or fewer days of light aerobic

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exercise per week. All subjects were asked to refrain from intense or unaccustomed exercise 24

hours prior to testing and to remain relatively inactive for the total duration of testing. In

addition, subjects were asked to abstain from caffeine on testing days as it has been shown to

affect muscular force at low activation frequencies (35).

Force measurements:

For isometric contractions of the thigh, subjects were seated in a custom-built force chair

with the hip and knee secured at approximately 70° of flexion. Both legs were firmly secured to a

rigid lever arm with an inelastic strap to ensure that the knee extensors could only perform

isometric contractions. The moment arm was established by placing a load cell (model 2000A;

Rice Lake Weighing Systems, West Coleman Street, Rice Lake, Wisconsin, USA) parallel to the

line of pull and perpendicular to the lever arm. Force was recorded from the load cell using a

MacLab A-D converter (model ML 400; ADInstruments, Milford, Mass., USA) sampling at

100 Hz and interfaced with a portable Macintosh computer (Apple Computer, Cupertino, Calif.,

USA). All force tracings were displayed and recorded on a computer.

Electrical stimulation (NMES). A high voltage electrical stimulation unit (Rich-Mar

Theratouch 4.7) was used to assess LFF and to evoke contractions during fatigue protocols.

Stimulation was delivered through two electrodes (8×10-cm) to evoke isometric contractions of

the quadriceps femoris muscle group. One electrode was placed 2-3 cm above the superior aspect

of the patella over the vastus medialis muscle. The second electrode was placed lateral to and 30

cm above the patella over the vastus lateralis muscle. Permanent ink marker was used to trace the

electrodes to ensure the same placement for the 24-hour assessment of LFF, or at any other time

the electrodes were taken off during testing.

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Force of the quadriceps femoris muscle group, elicited at 20 Hz (P20) and 100 Hz (P100)

was measured before, immediately after, and at 10, 20, and 60 min, as well as 2, 4, 6 and 24

hours following a fatigue protocol. At each time point, the quadriceps femoris muscle group was

given two 1-sec contractions at 20 Hz followed immediately by two 1-sec contractions at 100

Hz. These contractions were elicited to potentiate the muscle. Approximately 10 sec following

these four contractions, a 1-sec contraction at 20 Hz was given followed immediately by a 1-sec

contraction at 100 Hz for the assessment of LFF and these will be referred to as ‘evaluation

stimulations’. These 1-sec contractions at 20 and 100 Hz were elicited three times for each

assessment. The ratio of P20/P100 was calculated and the percentage reduction in this ratio was

used to evaluate LFF (7, 39). A greater percent reduction in P20/P100 from baseline values (pre-

fatigue) indicates greater LFF.

Fatigue tests for both SCI and able-bodied subjects were elicited via NMES. All

contractions during the fatigue tests were evoked with 30-Hz trains of 450-µs biphasic pulses

with a 33% duty cycle (3 s on/6 s off). The fatigue test was evoked in one thigh (experimental

leg). The contralateral, un-fatigued leg served as control. The experimental leg was assigned by

counterbalancing dominant/non-dominant leg for each subject. The measurements of P20 and

P100 were assessed in both thighs.

Experimental protocol:

Familiarization. Subjects were seated in the custom designed isometric force chair and


and two electrodes were placed on each thigh as previously described. Electrical stimulation at a

frequency of 100 Hz was given to evoke 1-3 sec isometric contractions of the knee extensors at

increasing current levels until 22.7 kg of isometric force was produced in able-bodied subjects. If

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this level of NMES current was tolerated well and subjects were still interested in participation,

they were asked to return to the laboratory 1-2 days later for testing. For SCI participants, the

current was increased until they could produce a minimal of 4.5 kg of isometric force with

maximum not exceeding 18 kg. Force in the SCI group was kept below 18 kg to substantially

reduce the risk of fracture when performing isometric contractions (12).

Test Session. Subjects were seated in the custom designed isometric force chair and their

legs and torso were firmly strapped into the chair. Subject’s legs were cleaned with alcohol and

two electrodes were placed on each thigh as previously described. Electrical stimulation

amplitude at 100 Hz was slowly increased over several 1-sec contractions to achieve ~18 to 22.7

kg of isometric force in each thigh for SCI and able-bodied subjects, respectively. After the

NMES amplitude was determined, it remained the same in each leg throughout the duration of

the test session for the measurements of P20 and P100, as well as for fatigue tests. As stated

previously, P20 and P100 were measured before, immediately after, and at 10, 20, and 60 min, as

well as 2, 4, 6 and 24 hours following an NMES-evoked fatigue test.

For this session, the NMES-evoked fatigue test for able-bodied individuals lasted 15 min

and consisted of 3-sec isometric contractions of the thigh with 6-sec rest between contractions

(33% duty cycle). This allowed for a total of one hundred 3-sec contractions, which were evoked

at the same NMES amplitude used for measurement of P20 and P100. All able-bodied subjects

were tested first to determine their average force loss during this fatigue protocol. The SCI group

performed a fatigue test that was terminated when mean force loss was equivalent to that of the

able-bodied group during the 15 min fatigue protocol. This was done in an effort to match

relative fatigue between groups. The duty cycle for the SCI fatigue test was the same as in able-

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bodied (33%; 3 sec on/6 sec off) and ranged between 2-8 min, which was dependent on

individual force reductions in the SCI group.

Assessment of muscle pain:

Muscle pain was evaluated in both the experimental and control leg based on a 10-point

Likert-type scale in the able-bodied subjects only. Participants were asked to rate the intensity of

leg muscle pain from 0 “no pain at all” to 10 “worst possible pain imaginable”. Subjects verbally

reported these pain ratings before, and at 24 and 48 hours following the NMES-evoked fatigue

test.

Analysis of force tracings:

Force tracings for the first and last three contractions of the fatigue protocol were

analyzed by measuring force-time integrals (FTI). Fatigue was calculated as follows:

Fatigue = [FTI (1st 3 contractions) – FTI (last 3 contractions)] ÷ FTI (1st 3 contractions)

Single contractions for the assessment of P20 and P100 were always 1 sec in duration. For each

force tracing at 20 and 100 Hz, the average value for the first 0.5 sec after the initial rise was

analyzed. The highest mean force at 20 and 100 Hz was used in the calculation of the P20/P100

ratio at each time point.

Statistical Analyses:

The SCI subject population in this study consisted of individuals with both complete and

incomplete spinal injuries. Because we found no prior statistically significant differences in LFF

results between complete and incomplete subjects, the entire SCI population was combined for

comparison with able-bodied subjects.

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Data analyses were performed using SPSS version 13.0 (SPSS Inc., Chicago, IL).

Independent t-tests were used to assess potential differences between the groups in subject age,

body mass, and percent force loss during NMES-evoked fatigue protocols. In the t-test analyses,

the homogeneity of variance was tested using Levene’s test for equality of variance. Statistics

were adjusted accordingly if the assumption of equality of variance was violated.

For all analyses, the ratio of the P20/P100 was calculated for each time point. These time

points were then reported, as the percentage reduction from baseline and from here on will

represent LFF. The magnitude of LFF will be assessed by the percentage reduction from baseline

P20/P100 values up to 1 hour following the fatigue protocol. This time period was selected

because it tests our first hypothesis about the magnitude of LFF after the fatigue protocol. The

recovery of LFF will be examined over the entire 24 hour post-fatigue period because prior

empirical research suggests that SCI would have muscle injury following NMES-evoked

isometric contractions (2), which can dramatically affect LFF (6).

The magnitude of LFF in the experimental leg was analyzed using a 2 (group: SCI, able-

bodied) x 4 (time: 0, 10, 20, and 60 minutes post-fatigue) mixed model ANOVA with a repeated

measure on the time variable. Magnitude of LFF was not examined in the control leg because it

did not perform a fatigue protocol.

A separate analysis for recovery of LFF was performed for both the experimental leg and

the control leg in each group. Recovery of LFF was analyzed using a 2 (group) x 8 (time: 0, 10,

20, 60 minutes; 2, 4, 6, and 24 hours) trend analysis in order to examine differences in the rate of

recovery between groups after the fatigue protocol. Since the recovery of LFF was significantly

different in the ‘control leg’ between groups, a secondary analysis was performed. This

assessment included a 2 (group) x 8 (time) trend analysis for the experimental leg with control

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leg LFF values used as covariates. This allowed us to determine what effect the evaluation

stimulations have on the LFF response above and beyond that caused by the fatigue protocol.

Perception of thigh muscle pain in able-bodied participants was assessed at 24 and 48

hours post-fatigue. Pain data were analyzed using a 1 (group) x 3 (time: Pre, 24 & 48 hours post-

fatigue) ANOVA with repeated measure on the time variable.

Studentized t-tests were used to assess potential differences in 100 Hz force between

baseline and 24-hour post-fatigue data within-groups. Separate t-tests were run for both the able-

bodied and SCI. Significant reductions in 100 Hz muscle force at 24 hours were used as an

indicator of muscle injury.

All mixed model analyses were based on the F-statistic. Effects sizes for F-statistics were

expressed as partial eta-squared (η2). The Greenhouse-Geisser epsilon (ε) was reported and

degrees of freedom were adjusted when the sphericity assumption was violated (ie. if Mauchly’s

test of sphericity was statistically significant at p < 0.05). The familywise error rate was

controlled using the Bonferroni adjustment when tests of simple effects were conducted.

Results

Participant Characteristics:

All SCI participants were non-ambulatory and consisted of those with complete and

incomplete spinal lesions ranging from C5 to T9. Mean duration of spinal injury was 13.6 ± 12.2

years. Four of the nine SCI participants had limited sensory function in their thighs and none had

enough sensation to rate thigh muscle pain. Three of these 4 individuals had limited voluntary

motor function in the thigh area and were able to extend one or both knees with maximal

isometric forces less than ~9 kg. Sedentary able-bodied subjects were performing one or fewer

days of light aerobic exercise (ie. walking) per week. Individual subject characteristics for the

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SCI group are shown in Table 4.1. Mean characteristics for SCI and able-bodied groups are

shown in Table 4.2. Spinal cord injured subjects were significantly older than able-bodied

subjects (F (16) = 5.61, p = 0.025) but no differences existed between groups for body mass (F

(16) = 0.303, p = 0.838). All subjects completed this study without incidence.

Force Reduction During NMES-Fatigue Protocols:

Mean percentage force loss during the fatigue tests was -50.3 ± 9.0 % for SCI and

–49.6 ± 9.5 % for able-bodied, which were not significantly different (F (16) = 0.010, p = 0.870).

Force loss during NMES-evoked fatigue protocols for groups are shown in Figure 4.1. The able-

bodied fatigue test lasted 15 minutes and consisted of 100 contractions (3 s on/ 6 s off). The SCI

fatigue test was performed at the same duty cycle (3 s on/ 6 s off) and lasted 4.3 ± 1.8 min, which

was dependent on individual force loss in this group. This protocol was used with the SCI group

to produce an equivalent force loss as the able-bodied group during the 15 min fatigue protocol

(~50%).

Force Tracings:

Representative force tracings at 20 Hz and 100 Hz are shown in Fig. 4.2 for one SCI

participant before and 1-hour post-fatigue. Using the ratio of these forces allowed for

quantification of LFF. One hour following the fatigue protocol, 100 Hz force has returned to near

baseline values, whereas 20 Hz force is still depressed substantially.

100 Hz Force:

Figure 4.3 shows percent change from baseline P100 values over 24 hours post-fatigue,

for both the experimental and control leg in SCI and able-bodied subjects. No statistical tests

were performed for these data. Return of 100 Hz force to baseline values shortly after exercise

can be used to assess recovery of metabolic fatigue. However, the SCI group appears to have

67

minimal recovery of 100 Hz force over the post-fatigue period, which would indicate that

additional factors (ie. muscle injury) are affecting the response.

Magnitude of LFF:

The magnitude of LFF, expressed as percent change from baseline P20/P100 ratio

values up to 1 hour post-fatigue, was examined between groups. In the experimental leg, the

analysis showed that a significant group x time interaction existed (F (3,16) = 8.599, p < 0.001,

η2 = 0.350) (Figure 4.4a). However, when tests for simple effects were performed and adjusted

for multiple comparisons, there were no significant differences noted at any time point.

Therefore, we concluded that no differences existed for the magnitude of LFF between groups.

Recovery of LFF:

Trend analysis was used to examine differences between groups in the recovery of LFF

across the 24-hour post-fatigue period in both the experimental and control leg. In the

experimental leg, the recovery of LFF was significantly faster in the able-bodied group compared

to SCI (Figure 4.4b). This was based on a significant group by trend interaction for the linear

recovery of LFF in the experimental leg over the 24-hour period between groups (F (7,128) =

4.91, p < 0.001, η2 = 0.212). Decomposition of the interaction showed that able-bodied recovered

from LFF in the experimental leg at an 18% greater rate than the SCI group (t = 5.68, p < 0.001),

which was based on differences in slopes for the linear trend between groups. In essence, the SCI

group had little or no recovery of LFF over 24 hours.

Similarly in the control leg, the recovery of LFF was significantly faster in the able-

bodied group compared to SCI (Figure 4.5). This was based on a significant group by trend

interaction for the linear recovery of LFF in the control leg over the 24-hour period between

groups (F (7,128) = 2.30, p = 0.031, η2 = 0.112). Decomposition of the interaction showed that

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able-bodied recovered from LFF in the control leg at a 12% greater rate than the SCI group (t =

3.95, p < 0.001), which was based on differences in slopes for the linear trend between groups.

Since significant group differences were found for the recovery of LFF in the control leg,

a secondary analysis was performed. When the experimental leg was statistically adjusted for

control leg LFF values, recovery was not different between able-bodied and SCI individuals

(Figure 4.6). This was based on a lack of significant group by trend interaction for the recovery

of LFF over the 24-hour period between groups (F (7,127) = 1.98, p = 0.063, η2 = 0.098). There

was a significant main effect for time (F (7,127) = 6.34, p < 0.001, η2 = 0.259) but not for group

(F (1,127) = 0.76, p = 0.386, η2 = 0.006) for the recovery of LFF.

Indicators of Muscle Injury:

Muscle pain ratings in the experimental leg of the able-bodied group at 24 and 48 hours

post-fatigue were 2.0 ± 1.1 and 1.1 ± 0.9, respectively, and there was a significant main effect for

time (F (1,8) = 12.90, p = 0.007). No pain was reported for the control leg at 24 or 48 hours post-

fatigue. All able-bodied subjects reported some muscle pain in the experimental leg 24 hours

following the fatigue test, which ranged from 1-4 (out of 10), indicating that minor amounts of

muscle injury may have occurred in this group.

Significant reductions in 100 Hz muscle force at 24 hours (from baseline values) were

used as an indicator of muscle injury. Force at 100 Hz in the experimental leg was significantly

lower (-30.8 ± 19.6 %) in the SCI group at the 24-hour time point compared to baseline values (p

< 0.001). However, 100 Hz force in the able-bodied group was not different at 24 hours (p =

0.354). This suggests that muscle injury was likely more severe in the SCI compared to the able-

bodied group.

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Discussion

We were unable to accept our first hypothesis, as our results indicate that no differences

were found between SCI and able-bodied subjects for the magnitude of LFF after a fatigue

protocol. This was an unexpected finding as we predicted that even with the same force loss, the

SCI group would have a greater metabolic impairment, which might cause the magnitude of LFF

to be greater in this group. In our companion paper, we showed that sedentary, able-bodied

individuals had a greater magnitude of LFF compared to endurance-trained subjects after fatigue

protocols matched for the total amount of fatigue. We predicted that the extreme disuse and

unloading of paralyzed muscle would cause the magnitude of LFF to be more severe than

normally loaded muscle, but our results did not support this hypothesis.

To our knowledge, no one has examined the differences in LFF between able-bodied and

SCI subjects. However, Shields et al. (31) examined reductions in low frequency force (ie. 20

Hz) in the soleus muscle of chronic (> 3 years post-injury) and acute SCI (< 5 weeks post-injury)

following a 3-minute bout of intermittent contractions. Immediately following exercise, 20 Hz

force was reduced by 75% in the chronic SCI but only 16% in acute SCI group. After 5 minutes

post-fatigue, 20 Hz force had completely recovered in the acute SCI groups but was still

suppressed nearly 60% in the chronically injured group. Direct comparisons to the Shields et al.

(31) study is difficult because of differences in the subject groups tested and because they caused

extremely different amounts of fatigue in these two groups upon examining LFF. A strength of

the current study is that we examined LFF after causing the same amount of fatigue in both

groups. It is possible that if the SCI group performed the same 15-minute fatigue protocol as the

able-bodied group, that the magnitude of LFF may have been different between groups.

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We were able to accept our second hypothesis that able-bodied subjects would recover

faster from LFF. Our results demonstrate that when fatigue was matched between groups, that

the recovery of LFF in the experimental leg of the SCI group was significantly slower (~18%)

and had essentially no recovery over the 24 hours post-fatigue period. In the control leg, the able-

bodied group showed small amounts of LFF that remained constant over the time of the

experiment whereas SCI showed a progressive appearance of LFF that nearly matched the LFF

in the experimental leg by 24 hours. However, when the experimental leg LFF was statistically

for LFF in the control leg, recovery of LFF was not different between groups (p = 0.064). This

suggests that a large portion of the recovery from LFF in the SCI group can be attributed to the

evaluation stimulations. In the control leg of SCI (Fig 4.5), each additional set of evaluation

stimulations caused a further reduction in the P20/P100 ratio. In the SCI experimental leg (Fig.

4.4b) however, it appeared there was a ‘basement effect’ and once the P20/P100 ratio was

suppressed ~50%, each additional set of evaluation stimulations did not cause a further reduction

in this ratio. Therefore, it appears that both the fatigue protocol and evaluation stimulations affect

LFF in the SCI group but their relative impact cannot be determined in the present study.

Regardless, our results show definitively that if NMES is used to contract paralyzed muscle

repeatedly (ie. fatigue test in experimental leg) or used periodically over time (ie. evaluation

stimulations in control leg) that LFF will be severe and the recovery will be minimal over 24

hours.

The proposed physiological mechanism for the almost non-existent recovery of LFF in

the SCI group is probably related to contraction-induced muscle injury. It has been shown in

several studies that LFF is more severe when prior muscle injury has occurred (6, 15, 22, 27).

For example, Child et al. (6) examined the recovery of LFF after a bout of eccentric contractions

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of the thigh, which were evoked by NMES and caused significant muscle pain and injury. They

showed that subjects had significant LFF up to 3 days following this type of exercise. Since the

origin of LFF has been attributed to disruption in the excitation-contraction coupling process,

disruption of myofibers with injury can impair calcium release/reuptake rates and affect

muscular force (4), most predominately at low activation frequencies (22).

Unloaded skeletal muscle, as in SCI, has been shown to be more susceptible to muscle

injury upon reloading (2, 17, 21, 24, 33, 37). Models of extreme unloading have consisted of

immobilization, hind limb suspension and space flight. A more severe model of unloading

occurs in those with paralysis, which results in complete inactivation of skeletal muscle below

the point of spinal lesion. A previous study from our laboratory showed that individuals with SCI

are more susceptible to muscle injury than able-bodied subjects (2). In a study by Bickel et al.

(2), T2-weighted magnetic resonance images were taken of the quadriceps femoris muscle group

prior to, and 3 days after 80 NMES-evoked isometric contractions of the thigh in both able-

bodied and SCI subjects. Three days post-exercise, there was a greater relative area of muscle

injured for the SCI group (25%) as compared to able-bodied (2%). In addition, they showed that

NMES evoked force was reduced by 22% three days post-exercise in SCI whereas able-bodied

force was not different from baseline values.

Although the current study did not quantify muscle injury, it likely played a role in the

almost non-existent recovery of LFF in the SCI group, which was most evident from the data

collected in the control leg. In the control leg, approximately ninety 1-sec contractions were

performed from pre- to 24 hours post-fatigue, which was more than were evoked in the study by

Bickel et al. (2). In addition to these ninety 1-sec contractions, the experimental leg performed a

fatigue protocol that consisted of at least nineteen 3-sec contractions (dependent on individual

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fatigue values). Based on the findings by Bickel et al. (2), it is probable that muscle injury

occurred in both the experimental and control leg of SCI. In addition, the SCI group had a

significant reduction (~31%) in 100 Hz force in the experimental leg at 24 hours post-fatigue,

which can be best explained by the long-lasting effects of muscle injury. Even more dramatic

was the 20 Hz force loss in the SCI group (experimental leg) at 24 hours, which equated to ~66%

reduction from baseline values. Although all able-bodied subjects reported some muscle pain 24

hours post-fatigue (2.0 out of 10), no reduction was observed in 100 Hz force at this time point.

Small amounts of muscle injury in the able-bodied group may have occurred but it did not affect

the recovery of LFF as severely as it did in the SCI group.

We hypothesize that LFF in individuals with SCI would be less pronounced with

consistent NMES training, possibly by reducing muscle injury. Sabatier et al. (30) used NMES

resistance training of the thigh muscles in individuals with SCI and showed increases in fatigue

resistance after 12 weeks of training. The subjects performed only 80 dynamic knee extensions

per week, which significantly improved muscle size but would not be expected to cause reduced

fatigability. These researchers proposed that as training progressed muscle injury occurring

during contractions was reduced and therefore caused less force loss during a bout of

contractions. This potential reduction in muscle injury with training in the SCI population would

likely reduce the amount of LFF. Experimental evidence to support this hypothesis comes from a

study demonstrating that 10 weeks of eccentric training in rats significantly reduced LFF after a

fatiguing bout of exercise (39). Future studies need to examine how NMES-evoked training in

paralyzed muscle might affect LFF.

In addition to muscle injury, fast-twitch muscle fibers have been reported to be more

susceptible to LFF (25, 27). Numerous studies have demonstrated “slow to fast” fiber conversion

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after 1-2 years of SCI with increased expression of myosin heavy chain IIa and IIx (3, 10), as

well as faster contraction speeds (9, 29). Muscle biopsy data from SCI subjects 2-11 years post-

injury indicate significantly fewer percentages of slow-twitch fibers compared to able-bodied

controls (18). Although not examined in this study, greater percentages of fast-twitch muscle

fibers in the SCI group may have partially contributed to the LFF observed in this group.

The SCI subject population in this study consisted of individuals with both complete and

incomplete spinal injuries. Because of potential differences in muscle function between

complete and incomplete SCI, it might be possible that LFF would differ as well. However, we

did not find any statistically significant differences between our complete and incomplete SCI

individuals for either magnitude or recovery of LFF. There was a trend for the incomplete SCI

individuals to have slightly less LFF and to show more recovery of LFF in both the experimental

and control leg. Because of our small sample sizes, we cannot exclude the possibility that

differences exist between these two SCI subgroups. Future studies with larger sample sizes will

be needed to test this hypothesis. Our study does show that regardless of the degree of sensory

and/or motor function in SCI, individuals who are non-ambulatory and are not loading the

paralyzed musculature on a daily basis will have LFF that can be severe and can last for at least

24 hours following use of NMES.

Several potential limitations exist for this study. First, it was not feasible to have subjects

remain in the force chair for 24 hours and therefore they had to re-enter the force chair several

times throughout the study, which may have increased variability of our force measures. To

counter this we tried to be as meticulous as possible by setting up subjects in the force chair the

same way each time they arrived and by using the non-fatigued leg as control. Secondly, surface

electrical stimulation was used and changes in the properties of the electrodes and/or the skin

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over the course of the study may have altered conductance of electrical current and generation of

muscular force. Subjects were asked to remain relatively inactive over the course of the day and

the electrodes were traced to ensure the same placement for the 24-hour assessment of LFF, or at

any other time the electrodes were taken off during testing. Third, this study used submaximal

electrical current to evoke muscular contractions of the quadriceps muscle and therefore only

small portions of this muscle group were likely recruited. For the SCI group, this was done to

keep forces relatively low in order to reduce the risk of fracture in this population (12). For the

able-bodied group, submaximal current was used mainly to limit the pain associated with its use

in individuals with normal sensory function. Future studies may want to stimulate smaller

muscles to maximize the chance of activating the entire muscle. Despite increased variability that

may have occurred due to testing methods, we feel that the differences observed between these

groups for recovery of LFF are indeed ‘real’ and the robustness of the response in the SCI group

is truly remarkable.

Conclusion

This study shows that the magnitude of LFF after a matched fatigue protocol is not

different between SCI and able-bodied subjects. Over the 24-hour post-fatigue period, LFF in

able-bodied subjects recovers toward baseline values whereas no recovery is observed in SCI.

More importantly, simply assessing LFF with NMES in paralyzed muscle causes a progressive

increase in LFF, and can account for a substantial portion of the LFF over time. We propose that

muscle injury is the main factor responsible for the incomplete recovery of LFF in the SCI group

and future studies need to test whether fatigue in SCI subjects actually represents the

accumulation of muscle injury rather than fatigue.

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Low frequency fatigue has clinical implications for those with SCI and various other

neuromuscular disorders. These patient populations may use low-frequency NMES daily for

rehabilitation to make weak or paralyzed muscles contract. In addition, many electrically

stimulated exercise modalities used in the SCI population are evoked by low frequency NMES.

From this study, it is apparent that once the paralyzed muscle is stimulated sufficiently, forces at

low frequencies will be substantially suppressed for at least 24 hours or longer, which may limit

rehabilitation and/or fitness goals.

References


2. Bickel CS, Slade JM, and Dudley GA. Long-term spinal cord injury increases susceptibility

to isometric contraction-induced muscle injury. Eur J Appl Physiol 91: 308-313, 2004. 3. Burnham R, Martin T, Stein R, Bell G, MacLean I, and Steadward R. Skeletal muscle

fibre type transformation following spinal cord injury. Spinal Cord 35: 86-91, 1997. 4. Byrd SK. Alterations in the sarcoplasmic reticulum: a possible link to exercise-induced

muscle damage. Med Sci Sports Exerc 24: 531-536, 1992. 5. Castro MJ, Apple DF, Jr., Hillegass EA, and Dudley GA. Influence of complete spinal

cord injury on skeletal muscle cross-sectional area within the first 6 months of injury. Eur J Appl Physiol Occup Physiol 80: 373-378, 1999.

6. Child RB, Brown SJ, Day SH, Saxton JM, and Donnelly AE. Manipulation of knee

extensor force using percutaneous electrical myostimulation during eccentric actions: effects on indices of muscle damage in humans. Int J Sports Med 19: 468-473, 1998.

7. Edwards RH, Hill DK, Jones DA, and Merton PA. Fatigue of long duration in human

skeletal muscle after exercise. J Physiol 272: 769-778, 1977. 8. Fryer MW, Owen VJ, Lamb GD, and Stephenson DG. Effects of creatine phosphate and


9. Gerrits HL, De Haan A, Hopman MT, van Der Woude LH, Jones DA, and Sargeant

AJ. Contractile properties of the quadriceps muscle in individuals with spinal cord injury. Muscle Nerve 22: 1249-1256, 1999.

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10. Gerrits HL, Hopman MT, Offringa C, Engelen BG, Sargeant AJ, Jones DA, and Haan

A. Variability in fibre properties in paralysed human quadriceps muscles and effects of training. Pflugers Arch 445: 734-740, 2003.

11. Hamada T, Hayashi T, Kimura T, Nakao K, and Moritani T. Electrical stimulation of

human lower extremities enhances energy consumption, carbohydrate oxidation, and whole body glucose uptake. J Appl Physiol 96: 911-916, 2004.

12. Hartkopp A, Murphy RJ, Mohr T, Kjaer M, and Biering-Sorensen F. Bone fracture

during electrical stimulation of the quadriceps in a spinal cord injured subject. Arch Phys Med Rehabil 79: 1133-1136, 1998.

13. Hillegass EA and Dudley GA. Surface electrical stimulation of skeletal muscle after spinal

cord injury. Spinal Cord 37: 251-257, 1999. 14. Jones DA, Howell S, Roussos C, and Edwards RH. Low-frequency fatigue in isolated

skeletal muscles and the effects of methylxanthines. Clin Sci (Lond) 63: 161-167, 1982. 15. Jones DA, Newham DJ, and Torgan C. Mechanical influences on long-lasting human



17. LeBlanc A, Lin C, Shackelford L, Sinitsyn V, Evans H, Belichenko O, Schenkman B,

Kozlovskaya I, Oganov V, Bakulin A, Hedrick T, and Feeback D. Muscle volume, MRI relaxation times (T2), and body composition after spaceflight. J Appl Physiol 89: 2158-2164, 2000.

18. Martin TP, Stein RB, Hoeppner PH, and Reid DC. Influence of electrical stimulation on

the morphological and metabolic properties of paralyzed muscle. J Appl Physiol 72: 1401-1406, 1992.

19. McCully KK, Authier B, Olive J, and Clark BJ, 3rd. Muscle fatigue: the role of

metabolism. Can J Appl Physiol 27: 70-82, 2002. 20. Miller RG, Giannini D, Milner-Brown HS, Layzer RB, Koretsky AP, Hooper D, and

Weiner MW. Effects of fatiguing exercise on high-energy phosphates, force, and EMG: evidence for three phases of recovery. Muscle Nerve 10: 810-821, 1987.

21. Mozdziak PE, Pulvermacher PM, and Schultz E. Muscle regeneration during hindlimb

unloading results in a reduction in muscle size after reloading. J Appl Physiol 91: 183-190, 2001.

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22. Newham DJ, Mills KR, Quigley BM, and Edwards RH. Pain and fatigue after concentric and eccentric muscle contractions. Clin Sci (Lond) 64: 55-62, 1983.

23. Olive JL, Slade JM, Dudley GA, and McCully KK. Blood flow and muscle fatigue in SCI

individuals during electrical stimulation. J Appl Physiol 94: 701-708, 2003. 24. Ploutz-Snyder LL, Tesch PA, Hather BM, and Dudley GA. Vulnerability to dysfunction

and muscle injury after unloading. Arch Phys Med Rehabil 77: 773-777, 1996. 25. Powers RK and Binder MD. Effects of low-frequency stimulation on the tension-frequency

relations of fast-twitch motor units in the cat. J Neurophysiol 66: 905-918, 1991. 26. Ratkevicius A, Skurvydas A, Povilonis E, Quistorff B, and Lexell J. Effects of

contraction duration on low-frequency fatigue in voluntary and electrically induced exercise of quadriceps muscle in humans. Eur J Appl Physiol Occup Physiol 77: 462-468, 1998.



28. Rijkelijkhuizen JM, de Ruiter CJ, Huijing PA, and de Haan A. Low-frequency fatigue,

post-tetanic potentiation and their interaction at different muscle lengths following eccentric exercise. J Exp Biol 208: 55-63, 2005.

29. Rochester L, Chandler CS, Johnson MA, Sutton RA, and Miller S. Influence of electrical

stimulation of the tibialis anterior muscle in paraplegic subjects. 1. Contractile properties. Paraplegia 33: 437-449, 1995.

30. Sabatier MJ, Stoner L, Mahoney ET, Black C, Elder C, Dudley GA, and McCully K.

Electrically stimulated resistance training in SCI individuals increases muscle fatigue resistance but not femoral artery size or blood flow. Spinal Cord 44: 227-233, 2006.

31. Shields RK, Law LF, Reiling B, Sass K, and Wilwert J. Effects of electrically induced

fatigue on the twitch and tetanus of paralyzed soleus muscle in humans. J Appl Physiol 82: 1499-1507, 1997.



33. Slade JM, Bickel CS, and Dudley GA. The effect of a repeat bout of exercise on muscle

injury in persons with spinal cord injury. Eur J Appl Physiol 92: 363-366, 2004. 34. Stokes MJ, Edwards RH, and Cooper RG. Effect of low frequency fatigue on human

muscle strength and fatigability during subsequent stimulated activity. Eur J Appl Physiol Occup Physiol 59: 278-283, 1989.

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habitual and nonhabitual caffeine consumers. J Appl Physiol 89: 1719-1724, 2000. 36. Vanderthommen M, Duteil S, Wary C, Raynaud JS, Leroy-Willig A, Crielaard JM, and

Carlier PG. A comparison of voluntary and electrically induced contractions by interleaved 1H- and 31P-NMRS in humans. J Appl Physiol 94: 1012-1024, 2003.

37. Warren GL, Stallone JL, Allen MR, and Bloomfield SA. Functional recovery of the

plantarflexor muscle group after hindlimb unloading in the rat. Eur J Appl Physiol 93: 130-138, 2004.

38. Westerblad H, Bruton JD, Allen DG, and Lannergren J. Functional significance of Ca2+

in long-lasting fatigue of skeletal muscle. Eur J Appl Physiol 83: 166-174, 2000. 39. Willems ME and Stauber WT. Fatigue and recovery at long and short muscle lengths after


79

Figure 4.1. Percent reduction in force-time integrals during fatigue protocols in spinal cord

injured and able-bodied subjects. Fatigue protocols differed in length between groups and were

designed to produce the same amount of fatigue. Values are Mean ± SD.

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Figure 4.2. Representative force tracings at 20 Hz and 100 Hz for one spinal cord injured

participant pre- and 1-hour post-fatigue.

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Figure 4.3 (a-b). 100 Hz force values (% initial) after a fatigue test for the experimental (a) and

control leg (b) in SCI and able-bodied over 24 hours post-fatigue. Return of 100 Hz force to

baseline values shortly after exercise can be used to assess recovery of metabolic fatigue.

Minimal recovery of 100 Hz force in the SCI group indicates that non-metabolic factors may be

responsible. No statistical analyses were performed for these data. Values are Mean ± SD.

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Figure 4.4 (a-b). (a) Magnitude of LFF was not different in the experimental leg of SCI and able-

bodied subjects over 1 hour post-fatigue. Values are Mean ± SD. (b) Recovery of LFF in the

experimental leg over the 24-hour post-fatigue period between SCI and able-bodied subjects. † p

< 0.001, group x trend interaction. Recovery was 18% faster in able-bodied based on differences

in slopes between groups.

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Figure 4.5. Recovery of LFF in the control leg over the 24-hour post-fatigue period in SCI and

able-bodied subjects. Values are Mean ± SD. † p = 0.031, group x trend interaction. Recovery

was 12% faster in able-bodied based on differences in slopes between groups.

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Figure 4.6. Recovery of LFF in the experimental leg when statistically adjusted for LFF in the

control leg of SCI and able-bodied subjects. Values are Mean ± SD. When LFF values in the

control leg were used as covariates, no difference in recovery rate was observed between groups

(p = 0.064; η2 = 0.098).

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CHAPTER V

SUMMARY & CONCLUSIONS

This research showed that ET had less LFF than SED even when total fatigue was

matched, suggesting that endurance trained muscle is more ‘protected’ from LFF. Endurance

trained muscle did not have faster recovery of LFF, but other factors such as muscle injury make

interpretations of the recovery difficult. For SCI, the initial magnitude of LFF was not different

from able-bodied subjects but the recovery was essentially non-existent in SCI. More

importantly, simply assessing LFF with electrical stimulation in paralyzed muscle causes a

progressive increase in LFF with each set of evaluation stimulations, and seems to account for a

substantial portion of the LFF. This study overwhelmingly shows that SCI are more susceptible

to LFF with the use of NMES and the response is likely dictated by muscle injury.

Since many able-bodied subjects do not use NMES to evoke muscular contractions,

clinical relevance of LFF is less important for these individuals. The initial LFF response

between these groups is more important from a physiological perspective and determining the

potential mechanisms for these differences may be important as most submaximal, voluntary

contractions are elicited at low frequencies. Although the current study was not designed to

cause muscle injury, small amounts likely occurred, as NMES is a novel way to facilitate

muscular contractions in individuals without neuromuscular disorders. Future studies in this area

need to utilize voluntary, isometric contractions to examine potential differences in LFF between

ET and SED individuals. The performance of voluntary contractions will almost ensure that no

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muscle injury will occur and any differences in LFF can be attributed solely to the metabolic

perturbations between groups.

Low frequency fatigue has clinical implications in the SCI population, who may use low-

frequency NMES daily for rehabilitation to make weak or paralyzed muscles contract. In

addition, many electrically stimulated exercise modalities used in the SCI population are evoked

by low frequency NMES, such as ambulation and cycling. From this study, it is apparent that

once the paralyzed muscle is sufficiently stimulated, that forces at low frequencies will be

substantially suppressed for at least 24 hours or longer, which may limit rehabilitation and/or

fitness goals.

This study may guide future research towards designing NMES protocols to limit fatigue

and muscle injury in those with SCI. The current study shows that when paralyzed muscle is

contracted with NMES, limited numbers of contractions should be evoked (to potentially limit

injury) and at least 2 days should be allowed for recovery before subsequent use of NMES. This

rest period will increase the chance of achieving adequate muscular force (especially at low

frequencies) to improve fitness, functional recovery from SCI, or whatever the patient’s goals

may be.

So what can be done to limit LFF, especially in the SCI population? The use of high

frequency NMES could offset the effects of low frequency fatigue. However, many rehabilitation

and exercise programs consist of repetitive muscular contractions and use of high frequency

stimulation over a single bout of contractions causes pronounced fatigue compared to lower

frequencies (3). Lieber and Kelly (3) used NMES to evoke contractions of the quadriceps muscle

at 10, 30, and 50 Hz for thirty minutes at a duty cycle of 50%. They demonstrated that increasing

frequency caused a greater decline in torque over time, which is well supported in the literature.

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If NMES were to be used to evoke repetitive, intermittent contractions, then the use of high

frequencies would not be feasible if muscular force is a goal of the rehabilitation/exercise

program.

We hypothesize that LFF in individuals with SCI would be less pronounced with

consistent NMES training, possibly by reducing the amount of muscle injury. Although

protection from a repeat bout of exercise has never been shown in individuals with SCI, it almost

certainly exists. If muscle damage were excessive each time NMES was used to contract

paralyzed muscle one would hypothesize that over time the muscle would actually get smaller

with training. This is not the case as NMES-induced resistance and endurance training cause

muscle hypertrophy in individuals with SCI (4, 7). Future studies need to examine how

subsequent bouts of NMES-evoked contractions influence muscle injury in the SCI population

and how training of the paralyzed musculature might affect LFF.

Studies need to test whether fatigue in SCI subjects actually represents the accumulation

of muscle injury rather than fatigue. Muscle fatigue has repeatedly been shown to be greater

during NMES-evoked contractions in SCI compared to able-bodied subjects and has been

attributed to increased proportions of fast-twitch fibers (7), reductions in oxidative enzymes (5),

and alterations in calcium handling (2, 8). However, these factors cannot fully contribute to the

enhanced fatigue, which can be several-fold greater in SCI subjects. Most of these studies

examine ‘fatigue’ during a bout of NMES-evoked contractions but do not mention muscle injury

as a potential mechanism for the response. A study by Castro et al. (2) was the first to mention

muscle injury as a potential explanation for altered muscle mechanics (relaxation time) after SCI.

Bickel et al. (1) followed up by showing that NMES-evoked contractions of the thigh caused

significant muscle injury in individuals with SCI. However, this study did not try to elucidate

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what factors caused the nearly two-fold greater force loss in SCI compared to able-bodied

individuals. It is evident that future research should try to determine what role muscle injury

plays on the ‘fatigue’ response in individuals with paralysis.

In the current study, LFF is likely attributed to muscular fatigue as well as injury.

However, it seems that it is not solely dictated by either of the two factors. Muscle fatigue is

defined as ‘a reduction in expected force output’ and is not due to structural changes occurring

within the muscle. With muscle injury there is also a reduction in expected force output but it is

due to structural disruption of the muscle, which can subsequently impair metabolic processes

(6). It is our contention that LFF meets somewhere along the Fatigue/Injury continuum, which is

shown in Figure 5.1.

For these reasons, the term ‘low frequency fatigue’ should possibly be replaced with a

more appropriate term. The word ‘fatigue’ in ‘low frequency fatigue’ would suggest that force is

reduced only because ionic/metabolic changes have occurred, but no structural alteration within

the muscle has occurred. In the current project, LFF probably does not exist completely devoid

of muscle injury and therefore in the future, the use of a term such as ‘low frequency force ratio’

may be more appropriate.

References

1. Bickel CS, Slade JM, and Dudley GA. Long-term spinal cord injury increases susceptibility to isometric contraction-induced muscle injury. Eur J Appl Physiol 91: 308-313, 2004.

2. Castro MJ, Apple DF, Jr., Rogers S, and Dudley GA. Influence of complete spinal cord

injury on skeletal muscle mechanics within the first 6 months of injury. Eur J Appl Physiol 81: 128-131, 2000.

3. Lieber RL and Kelly MJ. Torque history of electrically stimulated human quadriceps:

implications for stimulation therapy. J Orthop Res 11: 131-141, 1993. 4. Mahoney ET, Bickel CS, Elder C, Black C, Slade JM, Apple D, Jr., and Dudley GA.

Changes in skeletal muscle size and glucose tolerance with electrically stimulated resistance

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training in subjects with chronic spinal cord injury. Arch Phys Med Rehabil 86: 1502-1504, 2005.

5. Martin TP, Stein RB, Hoeppner PH, and Reid DC. Influence of electrical stimulation on

the morphological and metabolic properties of paralyzed muscle. J Appl Physiol 72: 1401-1406, 1992.

6. McCully KK, Argov Z, Boden BP, Brown RL, Bank WJ, and Chance B. Detection of

muscle injury in humans with 31-P magnetic resonance spectroscopy. Muscle Nerve 11: 212-216, 1988.

7. Mohr T, Andersen JL, Biering-Sorensen F, Galbo H, Bangsbo J, Wagner A, and Kjaer

M. Long-term adaptation to electrically induced cycle training in severe spinal cord injured individuals. Spinal Cord 35: 1-16, 1997.

8. Talmadge RJ, Castro MJ, Apple DF, Jr., and Dudley GA. Phenotypic adaptations in

human muscle fibers 6 and 24 wk after spinal cord injury. J Appl Physiol 92: 147-154, 2002.

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Figure 5.1. The mechanisms responsible for force loss along the Injury/Fatigue continuum.

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