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Delayed muscle soreness, muscle function and evidence of leukocytes in human skeletal muscle following… MacIntyre, Donna Lee 1994

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DELAYED MUSCLE SORENESS, MUSCLE FUNCTION AND EVIDENCE OFLEUKOCYTES IN HUMAN SKELETAL MUSCLE FOLLOWING ECCENTRICEXERCISEbyDONNA LEE MACINTYREDiploma in Physiotherapy, University of Alberta, 1970B.S.R.(P.T.), University of British Columbia, 1980M.P.E., University of British Columbia, 1986A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYInTHE FACULTY OF GRADUATE STUDIESInterdisciplinary StudiesWe accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIANovember, 1994© Donna Lee Maclntyre, 1994In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the library shall make itfreely available for reference and study. 1 further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)___________________________Department of reuL ?-ke.cLLThe University of British ColumbiaVancouver. CanadaDate 1.otDE-6 (2/88)11ABSTRACTThe three primary purposes of these studies were to 1) characterize the time courseand the relationships among delayed onset muscle soreness, eccentric muscle torque, serumcreatine phosphokinase and urinary hydroxyproline in response to two exercise durations;2) to examine whether there was increased presence of leukocytes in the exercised musclecompared to the contra-lateral non-exercised muscle; and 3) to determine the time courseand the relationships among eccentric muscle torque, muscle fatigue, range of motion, thepresence of leukocytes, and delayed onset muscle soreness following the eccentricexercise. In the first study, the dependent variables were measured before and after 180(shorter duration) and 300 (longer duration) eccentric repetitions of the quadriceps muscles.Eccentric torque of the quadriceps muscles was evaluated utilizing the same parameters ofrange of motion and velocity of movement as the exercise stimulus. Muscle soreness wasevaluated by the Descriptor Differential Scale which reflects the sensory (intensity ofsoreness) and affective (unpleasantness) components of the discomfort. In the secondstudy, a radionuclide technique was used to determine the presence of leukocytes in theexercised muscle. Muscle fatigue was assessed with a power spectrum analysis. Thegreatest intensity of soreness and unpleasantness was between 20 and 48 hours post-exercise. The presence of leukocytes in the exercised muscle was significantly greater thanin the contra-lateral non-exercised muscle. Additional data collection before 24 hours postexercise revealed a biphasic response in eccentric torque, not reported previously inhumans. Eccentric torque declined immediately after the exercise stimulus and again at 20to 24 hours post-exercise. Fatigue of the vastus lateralis muscle was most evident at 2hours post-exercise. The greatest loss of range of motion occurred at 24 hours postexercise. Significant correlations were found between unpleasantness and creatinephosphokinase, intensity of soreness and creatine phosphokinase, and unpleasantness and111range of motion. Using a radionuclide imaging technique allowed a quantitative evaluationof the widespread distribution of the leukocytes compared to the smaller sample that wouldhave been evaluated from microscopic examination of human muscle biopsies. Previouslyonly focal injury has been shown due to the sampling limitations of using isolated musclebiopsies.ivTABLE OF CONTENTSAbstract iiTable of Contents ivList of Tables viList of Figures viiiList of Abbreviations xAcknowledgements xiiDedication xiiiChapter One The Effects of Two Durations of Eccentric Exercise on DelayedMuscle Soreness, Muscle Strength, and Biochemical Markersof Muscle Injury 1Introduction 1Purpose 4Research Hypotheses 4Methods and Procedures 5Results 11Discussion 26Conclusions 33References 35Chapter Two The Effects of Eccentric Exercise on Delayed Muscle Soreness,Muscle Strength and Fatigue, Joint Range of Motion, and thePresence of Leukocytes in Exercised Muscle 40Introduction 40Purpose 43Research Hypotheses 43Methods and Procedures 44Results 51VDiscussion 75Conclusions 84References 86Chapter Three Concluding Remarks 91References 95Appendix One Review of the Literature 96References 104Appendix Two Descriptor Differential Scale 110Instructions to the Subjects 110Calculations 110Reliability and Validity 119References 123Appendix Three Power Spectrum Analysis 124Calculations 124Reliability and Validity 131Fatigue of the Rectus Femoris Muscle 134Fatigue of the Vastus Lateralis and VastusMedialis Muscles 141References 144Appendix Four Radio-isotope Investigation of Acute Inflammation 146Labelling Procedure 146Analysis of the Presence of Tc-99m WBC 147Possible Mechanisms Contributing to theResponses of Two Subjects 159References 178Appendix Five Individual Subject Data - Chapter One 180Individual Subject Data - Chapter Two 199viLIST OF TABLES1. The Effects of Eccentric Exercise on Muscle Soreness, Muscle Strengthand Biochemical Markers 122. Two Durations of Eccentric Exercise: Manova Tables 133. Two Durations of Eccentric Exercise: Post Hoc Contrasts 204. Relationships Among Muscle Soreness, Muscle Strength and BiochemicalMarkers at Each Test Time 215. Indirect Measures of Exercise-Induced Muscle Injury Before and AfterEccentric Exercise. 536. The Presence of White Blood Cells in Four Regions of Interest in theExercised Muscle After Eccentric Exercise 547. Manova Tables for the Indirect Measures 558. Manova Tables for the Presence of White Blood Cells in Four Regions ofInterest 569. Post Hoc Contrasts of the Indirect Measures 5710. The Presence of White Blood Cells in the Four Regions of Interest:Post Hoc Contrasts 7111. Relationships Among Muscle Soreness, Muscle Strength and Fatigue,and Range of Motion at Each Test Time 7212. Pearson Product-Moment Correlations for the Descriptor DifferentialScale 12113. Power Spectrum Analysis: Pearson Product-Moment Correlation Coefficients 13314. Power Spectrum Analysis: Descriptive Statistics of the Median Frequenciesof the Quadriceps Muscles 13815. Power Spectrum Analysis: Paired T-Tests 14316. Mean Count/Pixel for Each Region of Interest for Each Subject at EachTestTime 15117. Coefficient of Variation for the Regions of Interest 15418. Subtraction of the Background 15719. Technetium-99m White Blood Cells for Each Region of Interest at EachTest Time 16620. Normalized Data for Each Region of Interest 168viiviiiLIST OF FIGURES1. Muscle Soreness Before and After Two Durations of Eccentric Exercise 142. Eccentric Torque Before and After Two Durations of Eccentric Exercise 163. Biochemical Markers Before and After Two Durations of Eccentric Exercise 184. Regions of Interest for Radio-Isotope Scanning 495. Characteristics of Eccentric Exercise: Muscle Soreness 606. Characteristics of Eccentric Exercise: Eccentric Torque 627. Characteristics of Eccentric Exercise: Range of Motion of the Knee 648. The Presence of White Blood Cells in the Four Regions of Interest inthe Exercised Muscle 669. Slopes of the Median Frequencies of Two of the Quadriceps Muscles 6810. The Relationship Between Eccentric Torque and Vastus Lateralis MedianFrequency at Two Hours Post-Exercise 8111. Amount of Sensation 11112. Amount of Unpleasantness 11313. Subject 1: Intensity of Soreness at 24 Hours Post-Exercise 11514. Subject 1: Unpleasantness Response at 96 Hours Post-Exercise 11715. Force and Raw Electromyography (EMG) Signals 12516. Typical Surface-EMG Power Spectrum 12717. Regression Lines for the Frequency Spectra 12918. Power Spectrum Analysis: Rectus Femoris Median Frequency 13519. Slopes of the Median Frequencies of the Superficial Quadriceps Muscles 13920. Subject 4: Antero-Distal Region of Interest 148ixThe Presence of Technetium-99m White Blood Cells in the Four Regions ofInterest for Each Subject21. Subject 1 and Subject 2 16122. Subject 3 and Subject 4 16223. Subject 5 and Subject 6 16324. Subject 7 and Subject 8 16425. Subject 9 and Subject 10 165The Responses of Two Subjects Compared to the Group Mean ± OneStandard Deviation26. A. Range of Motion of the Right KneeB. Eccentric Torque 17327. A. Intensity of SorenessB. Unpleasantness 17428. A. Vastus Lateralis Median FrequencyB. Vastus Medialis Median Frequency 175xLIST OF ABBREVIATIONSADP adenosine diphosphateATP adenosine triphosphateCa calcium ionCMRR custom mode rejection ratioCPK creatine phosphokinaseCr creatimneCV coefficient of variationDDS Descriptor Differential ScaleDOMS delayed onset muscle sorenessECM extracellular matrixEMG electromyographyT-IIvIPAO (RR,SS)-4,8 diaza-3 ,6,6,9-tetramethyl undecane2, 10-dione bisoximeICC intraclass correlation coefficientLRP leukocyte rich plasmaMANOVA multivariate analysis of variance02 oxygenOHP hydroxyprolinePAF platelet activating factorPPP platelet poor plasmaRF rectus femorisRM repetition maximumROT regions of interestROM range of motionSD standard deviationTc-99m technetiumTc-PYP technetium pyrophosphateTNF tumor necrosis factorVAS visual analogue scaleVL vastus lateralisVM vastus medialisWBC white blood cellsxixiiACKNOWLEDGEMENTSI would like to extend my sincere appreciation to Dr. D.C. McKenzie, who was mysupervisor throughout the course of my PhD program. I am very grateful to him for hiswise counsel; his advice, support, and encouragement in helping me to complete my PhD.I am also grateful to my committee members - Dr. A.N. Belcastro, Dr. B.H. Bressler, Dr.P.W. Hochachka, Dr. D.M. Lyster, and Dr. W.D. Reid. They each provided me withideas and direction, and were generous in sharing their expertise.I would also like to thank Dr. Jonathan Berkowitz for advising me on the statisticalanalysis of my data. Dr. Michael Slawnych developed the computer program for the powerspectrum analysis. My thanks to him for his assistance and his advice regarding theelectromyography. I am also grateful to the staff of the Nuclear Medicine Departments atVancouver Hospital and at the University Site for their unending patience with me and theircooperation in scheduling my subjects. My thanks also to the staff in the ClinicalLaboratory at University Site and the Mineral Metabolism Laboratory at VancouverHospital. I am grateful to Mr. Shaffiq Rahemtulla who provided technical assistance withphotographs and slides. I also wish to thank Ms. Diana Jesperson, Mr. Randy Celebriniand Mr. Greg McGann for their assistance in collecting the data.I am indebted to British Columbia Health Research Foundation and CanadianFitness and Lifestyle Research Institute for their financial support of the studies thatcomprised my thesis.xiiiDEDICATIONThis thesis is dedicated to -my Mother, who taught me what is important in life;both of my parents, who have surrounded their children and grandchildren withunconditional love and support for whatever they have chosen to do;Roger, without whom I would not have completed my PhD. Thank you forthe adventures we’ve shared;my friends and colleagues, who in small ways and in substantial ways, helped mealong the way.My thanks to all of you.1CHAPTER ONETHE EFFECTS OF TWO DURATIONS OF ECCENTRIC EXERCISE ONDELAYED MUSCLE SORENESS, MUSCLE STRENGTH, AND BIOCHEMICALMARKERS OF MUSCLE INJURYIntroductionDelayed onset muscle soreness (DOMS) is a sensation of discomfort associatedwith movement or palpation usually felt in skeletal muscle 24 to 72 hours followingunaccustomed muscular exertion (1). The sensation of discomfort following overuse of amuscle can be very severe and appears to have characteristics similar to pain. Certaininvestigators have suggested that there is both a sensory and an affective component topain (2, 3). The sensory component refers to the physiological stimulation of theperipheral receptors and nerves but this sensory component is further modulated by theindividual’s past experience where attitudes and psychological variables may influencedescription of the sensation (4). By attending only to the sensory aspect of pain, thecomplete picture of the pain process is overlooked (5). As muscle soreness is a sensationof discomfort which may be associated with pain, it would seem appropriate to assessboth the intensity of muscle soreness (sensory component) and the related unpleasantness(affective component) associated with the sensation.Although discomfort may have more than one component, studies performed to dateexamining DOMS have reported assessment of the intensity of the sensation butconsideration of any affective component of DOMS has been neglected (6-10). Further,investigations examining DOMS have often used a visual analog scale (VAS) (7) or arating scale where a given number was chosen based on the corresponding adjective (6,29-11). When used for repeat testing, these scales may suffer from scaling error, whichincludes repeatedly using the same category or part of the line, or remembering a pastspecific response (2). This may limit the usefulness of the data derived, especially inextrapolation of the results to different populations. The Descriptor Differential Scale(DDS) enables collection of multiple responses and minimizes the scaling error.Although it is clear that DOMS results from overuse of muscle, especially as aresult of eccentric exercise, the specific etiology is not well understood. In 1990, Stauberand colleagues (12) suggested that DOMS was due to a complex set of reactionsinvolving disruption of connective tissue and the muscle fibre. Similar to Stauber andcolleagues, Smith (13) reported that mechanical disruption to the muscle fibre andconnective tissue is a result of the unaccustomed eccentric exercise, but the author alsosuggested that, as a result of the muscle injury, an acute inflammatory response begins.Similarities between DOMS and acute inflammation include pain, swelling and loss offunction as measured by muscle strength.Creatine phosphokinase (CPK) has been shown to increase after eccentric exercisebut it does not have the same time course as the perception of soreness (12, 14). Thedifferent time course may be related to the delay of the intracellular proteins entering theblood (12, 14), or the divergent response may be due to the location of the pain fibres inthe connective tissue and not in the muscle fibre (15).An early investigation by Abraham examined a marker of connective tissuedamage, the appearance of urine hydroxyproline (OHP), which was reported to be closelyrelated to the time course of DOMS (6). Abraham (6) also recommended that3standardizing the OHP excreted by the creatimne (Cr) excreted over 24 hours should givea more sensitive measure of the changes in Ol-IP.Studies which have related muscle force to DOMS have failed to find a closeassociation in the time course of these two parameters. However, in previous studies adifferent protocol was used to assess force compared to the protocol used to induce themuscle injury. Newham and colleagues (16) used maximum voluntary isometriccontractions of the knee extensors to evaluate strength but utilized a bench steppingexercise as the stimulus to induce DOMS. Fnden and colleagues (17) used isokinetic (atangular velocities of 180 and 300 degrees per second) and isometric strength to assessperformance following an eccentric bicycle exercise to elicit muscle soreness. In theabove mentioned studies, it is quite possible that different populations of muscle fibrescontributed to the performance tests compared to those affected by the initial bout ofexercise used to stimulate DOMS. A performance test more similar to the interventionused to induce the original muscle damage or muscle soreness would be a better choice toassess functional outcome.Tiidus and lanuzzo (18) have reported a lack of quantification regarding theamount of exercise required to produce DOMS or an increase in the activity of CPK.Subjects in their study performed concentric and eccentric knee extensor contractionsunder three different exercise regimes - intensity, duration and constant total work. Theirresults indicated that as exercise intensity increased (from 35% of 10 repetitionsmaximum [RM] to 90% of 10 RM through 150 repetitions) the sensation of musclesoreness increased. As well, long duration exercise (300 repetitions vs 100 or 200)produced elevated CPK levels and greater muscle soreness. More recently Newham andcolleagues (19) have reported that muscle soreness is greater when the muscle isexercised at a longer length compared to a shorter length.4In this study, both the sensory and affective components of DOMS were assessedby using the Descriptor Differential Scale (DDS). For each assessment of DOMS, thesubjects scored their intensity of soreness and unpleasantness immediately following thetest of eccentric torque. This standardized the experience for the subject’s assessment ofDOMS. In addition, the same parameters of exercise (position, range of motion, velocityof motion) were used to evaluate eccentric torque on subsequent days, as the eccentricexercise stimulus used to induce DOMS. Therefore the performance tests were likelyrecruiting a similar motoneuronal pool as the muscle fibres affected by DOMS. Andfinally, several parameters (DOMS, eccentric torque, CPK and OHP/Cr) were measuredin the same study.PurposeThe purpose of this study was to examine the time course and the relationships overtime among DOMS, muscle torque and indicators of muscle damage (CPK and OHP/Cr)in a sedentary group of individuals in response to two levels of eccentric exerciseduration.Research Hypotheses1. The magnitude of responses following the exercise stimulus will be directly related tothe duration of the exercise.2. The greatest perception of DOMS (measured as intensity of soreness andunpleasantness) will occur between 24 and 48 hours following the exercisestimulus, at the same time as the greatest OHP/Cr response, but earlier than thegreatest response in CPK.53. The timing of the greatest loss of eccentric torque will be different from the greatestperception of DOMS.Methods and ProceduresSubjectsTwenty individuals (16 females and 4 males) between the ages of 19 and 52 wererecruited into the study. However, the data from one subject was not included in theanalysis as she participated in another eccentric exercise activity (water skiing) during thesame week as data collection for this study. Individuals were excluded from the study ifthey engaged in recreational exercise of more than 4 hours/week, were running, joggingor lifting weights for the lower extremities, or involved in competitive sport. In addition,those individuals who may have had cardiovascular, neurological or musculoskeletalconditions that may have compromised their ability to perform the testing procedures orwhich may have posed a hazard to the individual as identified by their physician (ie. -cardiac disease, a chronic neurological disease, degenerative knee joint disease, a recentsoft tissue injury of the lower extremity) were excluded. Approval for this study wasgranted by the Clinical Screening Committee for Research Involving Human Subjects atthe University of British Columbia.ProtocolThe protocol was a randomized cross-over design, such that each of the subjectsperformed two different durations (shorter and longer) of the exercise stimulus in order to6induce DOMS. Following informed consent, subjects were randomly assigned to theorder of the exercise stimulus (shorter first or longer first). Subjects completed baselinemeasures (pre-test) of the DDS describing DOMS (intensity of soreness andunpleasantness), eccentric torque of the quadriceps, serum levels of CPK, and urine levelsof Ol-IP and Cr. This was followed by repetitive fatiguing eccentric contractions of thequadriceps at either shorter or longer duration (the exercise stimulus) on an isokineticdynamometer. The DDS, tests of muscle torque, and blood and urine samples wererepeated 24 hours (day 1), 48 hours (day 2), 96 hours (day 4), and 168 hours (day 7) afterthe exercise session. At least twelve weeks after the first testing session the protocol wasrepeated on the same leg in the same subject at the other duration. This interval waschosen because Evans found that repair of damaged muscle may take as long as twelveweeks (20).MethodsDescriptor Differential ScaleThe intensity of soreness and unpleasantness of muscle soreness was assessed usingthe DDS (2). This scale contains 12 descriptor items for each dimension assessed, andfor each item, the subject indicated if the intensity of soreness and then theunpleasantness was either equal in magnitude to that implied by each anchoringdescriptor or how much greater or lesser on a 21-point graphic scale (Appendix Two).For all subsequent assessments, subjects completed the scales immediately after a shortsession (the eccentric torque test) of a similar exercise that was used to initiate DOMS.To assess intensity of soreness, the subjects were instructed to evaluate DOMS elicitedthroughout the quadriceps muscles during the eccentric torque test. To assess7unpleasantness, they were asked to evaluate DOMS elicited throughout the quadricepsduring their daily activities.Gracely and Kwilosz (2) have reported the DDS to be reliable in the assessment ofpain between hours one and two (Pearson product-moment correlations of 0.82 forsensory intensity and 0.78 for unpleasantness) in a group of dental patients. SeeAppendix Two for reliability and validity of the DDS in subjects with DOMS.Eccentric/Concentric Torque of the Quadriceps MusclesThe subjects were seated on the KinCom (Medex Diagnostics of Canada,Coquitlam, B.C.) isokinetic dynamometer, with their hips at 80 degrees, their backsupported and the pelvis and thigh stabilized on the bench. The centre of rotation of theKinCom was positioned opposite the centre of the knee joint line. The resistance pad waspositioned distally against the tibia so that the lower edge of the pad was at a point on thelower leg that was 75% of the length of the fibula. The angular velocity was set at 30degrees/second through a range of 60 degrees at a long muscle length (110 - 50 degreesof knee flexion).The subjects performed three submaximal (the subjects were instructed to pushagainst the pad at approximately 50% of their maximal effort) and one maximal (thesubjects were instructed to push against the pad as hard as they could) practicecontractions, followed by four maximal contractions. A two minute rest was interposedbetween the warm-up contractions and the four maximal repetitions. The data wascollected during the last four maximal contractions, saved onto disk and subsequentlyanalyzed as the average eccentric torque of the knee extensors over repetitions two tofour.8Farrell and Richards (21) have reported the measurements of the KinCom system tobe repeatable (repeated loading and unloading of a strain gauge) and accurate to knownweights (Intraclass Correlation Coefficient [I.C.C.] = 0.99) in static testing. Duringdynamic testing, the applied force from trial to trial resulted in an I.C.C. of 0.95.Reliability of concentric and eccentric torque measurements (I.C.C.) on the KinCom havebeen reported to range from 0.93-0.98 for both slow (30 degrees/second) and fast speeds(180 degrees/second) in groups of healthy active subjects (22,23).The Exercise StimulusSubjects were seated on the KinCom as described above and performed repeatedeccentric contractions (110-50 degrees of flexion) at a slow speed (30 degrees/second)using the passive mode of the machine. The subjects were instructed to lift their legduring the concentric movement of the lever arm but not to push against the lever arm sothat production of lactic acid would be limited (14). When the machine changeddirection they were instructed to resist the eccentric movement of the lever armmaximally throughout the range of motion. Subjects had continuous visual feedback oftheir force from the computer screen. In the shorter exercise stimulus the subjectscompleted 180 repetitions - 18 sets of 10 repetitions of maximal eccentric contractions/setwith a one minute rest between each set. For the longer exercise stimulus subjectscompleted 300 maximal eccentric contractions - 30 sets of 10 repetitions with each setbeginning every minute for 30 minutes. This allowed a 20 second rest between each set.9Analysis of Creatine PhosphokinaseVenipuncture was performed at the time intervals noted in the protocol. The bloodsamples were immediately taken to the laboratory where they were centrifuged. Elevenmicrolitres (pL) of the specimen were placed on a Kodak Ektachem clinical chemistryslide (Eastman Kodak Co., ROchester, N.Y.). This analysis uses creatine phosphate andadenosine diphosphate as substrates to generate creatine adenosine triphosphate. In acoupled reaction sequence, hydrogen peroxide (which is produced in stoichiometricequivalents to ATP in the initial reaction) oxidizes a dye precursor. The rate ofchromophore production is monitored by reflectance spectrophotometry at 670 nm andused to measure CPK activity.Analysis of CreatinineTen mL of the 24 hour urine collection were sampled and taken to the laboratory foranalysis. In the laboratory, 10 j%L of the urine specimen were deposited on a KodakEktachem clinical chemistry slide. Creatinine diffused to gel layers where it washydrolyzed to creatine. During the initial reaction phase, endogenous creatine wasoxidized by reagents in the slide. Creatine was converted to sarcosine and urea bycreatine amidinohydrolase. The sarcosine was oxidized to glycine, formaldehyde andhydrogen peroxide. Rate determination was made at 3.85 and five minutes. The rate ofchange between the two readings was proportional to the creatimne concentration in thesample.10Analysis of HydroxyprolineSubjects were informed of the importance of a diet low in gelatin content in order tominimize the influence of endogenous sources of Ol-IP. Urine samples were collectedover 24 hours and refrigerated during the collection period. After thorough mixing, one 8mL aliquot was sampled and stored at -70 degrees centrigrade until the analysis wasperformed.Urine was assayed using the technique developed by Hughes and co-workers (24).In this method, OHP is derivatized with 4-chloro-7-nitrobenzofurazan, with subsequentestimation by reversed phase “high performance” liquid chromatography.Statistical AnalysisA two-way multivariate analysis of variance (MANOVA) for grouping factors(exercise duration) and repeated measures over time (24, 48, 96, 168 hours) was used oneach of the dependent variables (DDS intensity of soreness score, DDS unpleasantnessscore, eccentric torque, CPK, OHP/Cr) to examine for differences over time and betweenexercise durations. Post hoc analyses examined contrasts at each test time compared tothe pre-test and between test times. Only those variables that were significant over timewere examined as the multivariate F test is more robust than the univariate post hoc tests(25). Correlation analyses (Pearson product-moment correlation) were used to examinethe relationships among DOMS, eccentric torque of the quadriceps, CPK, and OHP!Cr.The significance levels were set at p < 0.05.11ResultsThere were no significant interactions and no significant differences betweenexercise durations (180 repetitions vs 300 repetitions) for any of the dependent variables(Table 2). However, intensity of soreness, unpleasantness and eccentric torque were allsignficantly different over time (p<0.001). The greatest intensity of soreness was at 24 to48 hours post-exercise (Figure 1A), while the greatest unpleasantness was at 48 hourspost-exercise (Figure 1B). The greatest loss of eccentric torque was at 24 hours post-exercise (Figure 2). There were no significant differences between exercise durations orover time for either CPK or OHP (Figure 3). The means and standard deviations (SD) foreach of the dependent variables for both exercise durations over time are presented inTable 1.Table 3 summarizes the post hoc contrasts for the dependent variables. Intensityof soreness (Figure 1A) and unpleasantness responses (Figure 1B) were all significantlyhigher than the pre-test, except for the final testing time at 168 hours (7 days) when boththe intensity of soreness and unpleasantness responses had returned to baseline levels.Between test time periods, only the intensity of soreness responses at 24 and 48 hourswere not significantly different. Eccentric torque at 24 and 48 hours were significantlylower than the pre-test (Figure 2). When contrasting the eccentric torque responsesbetween testing times only the 24 and 48 hour responses were not significantly different,similar to the analysis of the intensity of soreness.Correlation matrices (Table 4) revealed that the unpleasantness score and the CPKresponse, at 168 hours (7 days) after the shorter exercise duration, had the highestcorrelation at 0.90 (rZ=0.81). Other significant correlations (p<0.05) ranged from 0.51 -0.76 between CPK and intensity of soreness from 96 hours to 168 hours after the shorterTABLE1THEEFFECTSOFECCENTRICEXERCISEONMUSCLESORENESS,MUSCLESTRENGTHANDBIOCHEMICALMARKERSPre-test24Hours48Hours96Hours168HoursShorterexercisestimulus(180repetitions)IntensityofSoreness-7.8±4.0Unpleasantness-9.7±0.5Torque(N.m)121.7±46.3CPK*(lU/l)71.4±38.9Ol-IPt(ptmol/l)141.4±114.2Creatinine(jtmol/l)7293.9±833.4OHP/Cr18.8±•8.4±6.0-0.4±6.384.9269.2±203.5183.2±142.310745.6±6237.816.0±•10.1±5.62.1±•87.5±30.1181.3±134.8162.9±140.78941.0±79.417.4±9.02.9±8.5-4.4±5.3109±41.6216.4±253.2138.9±81.78384.3±4462.616.3±53-6.2±6.5-8.5±2.8125.4±48.4397.8±686.9143.1±87.99233.6±4831.815.7±3Longerexercisestimulus(300repetitions)IntensityofSoreness-8.1±2.9Unpleasantness-9.7±0.9Torque(N.m)114.4±•1CPK(lU/l)62.4±1.3OHP(mol/l)156.4+77.1Creatimne(umol/l)8870.0±3523.5OHP/Cr18.7±7.69.6±4.3-0.5±487.9335.2155.4±107.810222.1±6042.814.2±4.68.9±6.33.3±6.689.7496.7±1077.3136.7±84.88532.6±4592.916.2±7.43.5±8.4-3.3±6.6104.9±44.61321.9±4106.5163±103.28660.2±3324.017.4±7.0-8.5±1.8-9.5±0.8120.8365.6±670.8132.9±•18350.2±3627.315.3±8.7DataaremeansSDfor19subjects.*CreatinePhosphokinase,t=Hydroxyproline,=Hydroxyproline/CreatimneI- I)TABLE2TWO DURATIONS OF ECCENTRIC EXERCISE:MANOVA TABLESSource (of Variation) df MS FIntensity of SorenessWithin Cells 18 40.91Between Durations 1 11.90 0.29Within Subjects 72 27.85Time 4 2766.47 99•34*Within Cells 72 19.88Duration xTime 4 17.33 0.87UnpleasantnessWithin Cells 18 38.81Between Durations 1 2.55 0.07Within Subjects 72 18.38Time 4 1096.20 59.64*Within Cells 72 9.73Duration xTime 4 7.54 0.78Eccentric TorqueWithin Cells 18 617.00Between Durations 1 219.00 0.35Within Subjects 72 488.90Time 4 10605.00 21.69*Within Cells 72 343.05Duration xTime 4 196.61 0.57Creatine PhosphokinaseWithin Cells 18 3471457.4Between Durations 1 3972740.4 1.14Within Subjects 72 1493968.9Time 4 2440390.2 1.63Within Cells 72 1344252.7Duration xTime 4 2159078.8 1.61HydroxyprolineWithin Cells 18 6606.97BetweenDurations 1 1197.52 0.18Within Subjects 72 4908.27Time 4 4825.30 0.98Within Cells 72 5395.84Duration xTime 4 5332.74 0.99* p<0.o11314Figure 1. Muscle Soreness Before and After Two Durations of Eccentric Exercise.Values are means ± SD. There were significant differences over time p<O.OO1.* Significantly different from the pre-test p<O.OO1. + Significantly different thistest to previous test p<O.OO1.A. Intensity of SorenessB. UnpleasantnessOn all graphs where there is a pre-test, pre = pre-test.r\)I,,0 C ci)0HOD0 C U)0)0,C-U, CDMEANSCORE00(i1riiI-‘ CD-I,-a.-S CDMEANSCOREI--U,U,U,I..-i....+1FF[±rnHFFI-.C.,,16Figure 2. Eccentric Torque Before and After Two Durations of Eccentric Exercise.Values are means ± SD. There were significant differences over time p<O.OO1.* Significantly different from the pre-test p<O.OO 1. + Significantly different thistest to previous test p<O.O 1.ECCENTRICTORQUE(Nm)—-Ni-ODNiC000000‘T_Y—-s CD F’.)-o CD ccH m z 0 ccc+---- +I18Figure 3. Biochemical Markers Before and After Two Durations of Eccentric Exercise.Values are means ± SD.A. Creatine Phosphokinase (CPK)B. HydroxyprolineC. CreatinineD. The hydroxyproline/creatimne ratioAFigure3CI—200016001200a C) z <800r400 0350c:300250200a7,Pre2448--I--SHORTER•LONGER96B168TIME(Hours)2.000i01.52510E w 1.050104.57501000 30 25-200 x15C) a10-0•IF:•____+ Pre2448---SHORTEI]•LONGERII•I•-•——•—i•-—--•--Pre244896168TIME(Hours)D-IcIIIIIIt -----SHORTER•LONGER•_......4....•4•.•L_....•._I96TIME(Hours)168Pre244896TIME(Hours)I—168I—.cc20TABLE3TWO DURATIONS OF ECCENTRIC EXERCISE:POST HOC CONTRASTSSource Umvariate F pIntensity of Soreness df 1,18pre - 24 hr 125.80 <.001*pre-48hr 313.77 <.001*pre-96hr 41.60 <.001*pre-168hr 0.28 0.6024hr-48hr 0.62 0.4448 hr-96 hr 24.00 <.001*96hr-168hr 60.82 <.001*Unpleasantness df 1,18pre-24hr 69.75 <.001*pre-48hr 111.84 <.001*pre-96hr 32.47 <.001*pre-168hr 3.50 0.0724hr-48hr 16.69 0.001*48hr-96hr 52.55 <.001*96 hr - 168 hr 33.64 <.001*Eccentric Torque df 1,18pre-24hr 32.79 <.001*pre-48hr 23.59 <.001*pre -96 hr 4.02 0.06pre- 168 hr 1.01 0.3324hr-48hr 0.38 0.5448hr-96hr 13.70 0.002*96 hr - 168 hr 23.48 <.001**signifijnt differences between tests21TABLE 4RELATIONSHIPS AMONG MUSCLE SORENESS, MUSCLE STRENGTH ANDBIOCHEMICAL MARKERS AT EACH TEST TIMECORRELATION MATRICESShorter Exercise Stimulus (180 repetitions)Test Times (hours)Pre-test 24 48 96 168Intensity of SorenessTest Times (hours)UnpleasantnessPre-test 0.1324 0.72*48 0.54*96 0.81*168 0.93*Eccentric TorquePre-test 0.1824 -0.1148 0.3096 0.34168 -0.18Creatine PhosphokinasePre-test 0.0124 0.3448 0.3996 0.51*168 0.76*HydroxyprolinePre-test 0.2324 -0.0448 0.1896 0.03168 -0.07UnpleasantnessEccentric TorquePre-test -0.4224 -0.1648 0.1896 0.19168 -0.2222Shorter Exercise Stimulus cont.Test Times (hours)Pre-test 24 48 96 168Test Times (hours)Unpleasantness cont.pretestCatisPh011e480.35961680.360.52*pretest0xne0.90 *0.160.1696 -0.06168 -0.18Eccentric Torque0.14961680.15-0.03-0.140.23961680.380.13-0.09Creatine Phosphokinase-0.15961680.10-0.16Creatine-0.10Pre-test240.300.06961680.090.01-0.1023Shorter Exercise Stimulus cont.Test Times (hours)Pre-test 24 48 96 168HydroxyprolineTest Times (hours)CreatimnePre-test 0.73*24 0.74*48 0.79*96 0.89*168 0.57**= p<0.05Longer Exercise Stimulus (300 repetitions)Test Times (hours)Pre-test 24 48 96 168Intensity of SorenessTest Times (hours)UnpleasantnessPre-test 0.67*24 0.63*48 0.52*96 0.76*168 0.51*Eccentric TorquePre-test 0.0424 0.1548 0.2096 0.1768-0.18Creatine PhosphokinasePre-test 0.2024 0.2948 0.1196 0.34168 0.58*HydroxyprolinePre-test 0.1324 0.1248 0.2596 0.17168-0.3124Longer Exercise Stimulus cont.Pre-test 24 48 96 168UnpleasantnessTest Times (hours)Eccentric TorquePre-test -0.1824 -0.0848 -0.0496 -0.19168 -0.08Creatine PhosphokinasePre-test 0.0524 0.2348 0.3996 0.10168-0.10HydroxyprolinePre-test -0.0624 -0.0248 0.1796 0.42168-0.16Eccentric TorqueCreatine PhosphokinasePre-test 0.4524 0.2148 0.1896 0.01168 0.04HydroxyprolinePre-test 0.0224 0.2648 0.1396-0.04168-0.01Creatine PhosphokinaseHydroxyprolinePre-test -0.0624 -0.3048-0.0496-0.08168-0.20Test Times (hours)25Longer Exercise Stimulus cont.Test Times (hours)Pre-test 24 48 96 168Test Times (hours)Creatine Phosphokinase cont.CreatininePre-test 0.4348-0.150.271680.010.04CreatinineHydroxyprolinePre-test 0.57*0.83*960.60*1680.85*0.65**= p<0.0526exercise stimulus, and 0.58 at 168 hours after the longer exercise stimulus. RegardingCPK and unpleasantness, significant correlations ranged from 0.52 - 0.90 at 96 and 168hours after the shorter exercise stimulus. Other significant correlations were foundbetween the intensity of soreness and unpleasantness from 24 hours post-exercise to 168hours post-exercise for the shorter exercise stimulus, in which the correlations rangedfrom 0.54 to 0.93. The correlations between intensity of soreness and unpleansantnessafter the longer exercise ranged from 0.51 to 0.76 and were significant at all test times.There were also significant correlations between OHP and Cr at all test times over bothexercise durations. There were no other significant correlations among the dependentvariables.DiscussionBecause Tiidus and lanuzzo (18) found that intensity and duration of exerciseaffect both post-exercise serum enzyme activities and delayed muscle soreness, one of theaims of this study was to determine if there was a difference in outcome measuresbetween two durations of exercise. Even though the number of repetitions of the shorterexercise stimulus (180) was 60% that of the longer exercise stimulus (300 repetitions),and the rest periods were longer between sets during the shorter exercise, the outcomemeasures were not significantly different between the two exercise durations. In fact theywere essentially the same for all of the dependent variables except CPK at 48 and 96hours. But despite the large differences between the exercise durations for CPK, theextreme variability between subjects eliminated the possibility of finding significantdifferences (Table 1, Figure 3).27Tiidus and lanuzzo (18) reported their highest CPK response at 24 hours; beforethe highest soreness response at 48 hours. In this study the highest CPK responses wereafter the highest soreness responses. However, there were some differences between theprotocols of this study and that of Tiidus and lanuzzo. In this study the resistedquadriceps exercise was eccentric only, while Tiidus and lanuzzo utilized a concentricand eccentric protocol. Their subjects worked at 70% of 10 RM but in this study thesubjects were encouraged to give their best effort throughout the exercise. However,because of visual feedback of the force during the exercise stimulus, it was noted that theforce decreased as the exercise progressed in this study. Clarkson and colleagues (10)have proposed that different exercise regimes may result in different mechanismsproducing muscle soreness.All of the intensity of soreness responses from 24 hours to 96 hours weresignificantly higher than the pre-test with the greatest soreness at 48 hours after theshorter exercise and at 24 hours after the longer exercise (Figure 1A). By 168 hour (7days) the intensity of soreness had returned to the baseline. The perception ofunpleasantness was greatest at 48 hours after both exercise durations (Figure 1B). Theresponses at all testing times were significantly higher from the pre-test except theresponse at 168 hours (7 days) when the perception of unpleasantness had returned tobaseline. Sore muscles are often described as stiff, aching or tender (1, 26); sensationsthat are not only associated with intensity of discomfort. Individuals may also have anaffective, or emotional, response to the soreness. In assessing the sensation of pain,Meizack (5) has suggested that by only attending to one dimension the complete pictureis overlooked. The DDS is one scale that measures both the sensory and affectivecomponents of sensation.28However, the results of this study suggest that either unpleasantness (the affectivedimension of sensation) was not a prominent experience for these subjects or that theDDS differentiated between the two domains of discomfort. The highest unpleasantnessmean was 3.3 while the highest intensity of soreness mean was 10.1, indicating thatoverall the perception of unpleasantness was less than the perception of the intensity ofsoreness (Figure 1). Because muscle soreness after exercise is such a commonexperience, the subjects may have been less bothered by the unpleasantness of thediscomfort knowing that it would soon subside. Further study might compare theintensity of soreness and unpleasantness responses of healthy subjects with DOMS toindividuals who are beginning exercise after a lengthy immobilization and whoseresponse to a novel exercise has not been extensively reported. It may be that duration orintensity of soreness that is not predictable might have a greater affective component. Or,it could be that the affective dimension is not a prominent experience in muscle sorenesscompared to pain syndromes.The greatest decline in eccentric torque was at 24 hours after both exerciseintensities, although responses at 24 and 48 hours were both significantly lower than thepre-test (Figure 2). Other investigators have reported that the greatest decline in musclestrength is immediately after the exercise stimulus followed by a slow recovery for aweek or longer (10, 27). In this study, eccentric torque data was not collectedimmediately after the fatiguing exercise but it did take 96 hours (4 days) for torque toreturn to pre-exercise levels (Figure 2).It is interesting to note that intensity of soreness was highest and eccentric torquewas lowest between 24 and 48 hours. Figures 1A and 2 illustrate an extended time periodover which the greatest responses occurred. However, similar to other reports in theliterature there were no significant correlations between eccentric torque and the intensity29of muscle soreness (28). As suggested by Faulkner and colleagues (29), it may be thatthere is more than one mechanism contributing to the events in muscle after strenuouseccentric exercise. Friden and colleagues (17) have reported Z-line broadening andstreaming in the muscles of human subjects immediately after eccentric exercise. Thisdamage to the muscle may explain the loss of muscle strength that has been observedimmediately following eccentric exercise. Smith (13) has suggested that the sensation ofmuscle soreness evident between 24 and 48 hours post-exercise may be associated withan acute inflammatory response. Thus, first a mechanical injury and then a biochemicalresponse may explain the functional responses of the muscle after eccentric exercise.Nosaka and colleagues (30) have reported that eccentric exercise produces thelargest increase in CPK and a different time course from other types of exercise.Typically CPK does not begin to increase until 24-48 hours after eccentric exercise andreaches peak values three to six days after the exercise (30). Once again, mode ofexercise could explain the differences in the results of this study and those of Tiidus andlanuzzo (18) who observed a peak CPK response at 24 hours with a concentric/eccentricexercise routine. In this study, the CPK response after the shorter exercise stimulusappeared biphasic with the first increase at 24 hours and the greatest response at 168hours (7 days). After the longer exercise stimulus, CPK increased up to 96 hours (4 days)and then decreased at 168 hours (7 days)[Figure 3A]. Nosaka and colleagues (30), havesuggested that the delay in the rise of CPK may be due to a slowing of the transport ofenzymes through the lymph due to swelling and connective tissue damage. Clarkson andTremblay (31) proposed that the post-eccentric exercise appearance of CPK in the bloodis an indication of the onset of necrosis. At present there is no definitive explanation forthis long delay.30The characteristic intersubject variability of the CPK response after exercise hasbeen observed by others (10,32). Clarkson and colleagues (10) have suggested threegroupings of CPK response. High responders are those individuals with a peak CPKresponse over 2000 lU/L; medium responders have a peak between 500 and 2000 lU/Land low responders are those with a peak CPK response of less than 500 lU/L. Clarksonand colleagues (10) also compared the CPK responses in each of the groups to otheroutcome measures such as muscle soreness, isometric strength and joint angle. Theyconcluded that low CPK responders have smaller changes in the other measures as well.In this study there were not enough subjects to analyze the outcome measures accordingto these groupings. However, the CPK data was examined for extreme scores andanalyzed without the data from the three subjects who had peak CPK responses above2000 lU/L. The pattern of the responses over time was still the same as the full data set.Most importantly the pattern of responses was similar to what has been reported in theliterature (30). The greatest responses in CPK were observed from four to seven dayspost-eccentric exercise.Similar to CPK, the responses of OHP after strenuous exercise (Figure 3B) werenot significantly different between durations or over time. In attempting to determine themechanism underlying delayed soreness, Abraham first reported upon the presence ofOHP after a step-exercise (6). He suggested that changes in connective tissue metabolismcould be observed by monitoring changes in OHP excretion. Elevated urinary excretionof OHP following exercise may be interpreted as increased catabolism of collagen, amajor constituent of connective tissue (15,33). Abraham (6) recommended that OHP bereported relative to Cr because 90% of OHP is rapidly metabolized. In addition, thekidneys efficiently reabsorb the OHP excreting only approximately 10% into the urine.Because Cr excretion may be a reflection of kidney filtration, standardizing the Ol-IP31excreted relative to Cr excreted over 24 hours would give a more sensitive measure ofchanges in OHP metabolism.Even though Abraham found no significant increase in OHP or OHP/Cr, he founda significant correlation between OHP/Cr and soreness (6). From these results, heproposed that exercise induced muscle soreness may be related to disruption of theconnective tissue in the muscle.Stauber (34) suggested that the OHPICr ratio is only useful to standardize forkidney filtration under normal conditions, but in abnormal situations such as after kidneydamage, pregnancy, and muscle injury the relationship between Ol-IP and Cr changessubstantially. Considerable muscle damage can cause Cr release from muscle andelevated Cr levels in the urine. Therefore, in this situation OHP and Cr data should beexamined separately. Figure 3B and 3C illustrates the OHP data and Cr datarespectively. After the shorter exercise stimulus, the pattern of the Cr response was verysimilar to the shorter exercise OHP response. In fact, significant correlations were foundbetween OHP and Cr at all testing times (Table 4) supporting Stauber’s hypothesis that Cris released from damaged muscle. After the longer exercise stimulus Cr was highest at 24hours, a response which was not similar to OHP or CPK both of which were high at 96hours. However, significant correlations still existed at all testing times between OHPand Cr (Table 4). There were no significant correlations between CPK and Cr (Table 4).Figure 3D illustrates the OHP/Cr ratio. Just as Stauber had predicted, the Cr levels roseto a greater extent than OHP because the OHP/Cr ratio declined at 24 hours and eventhough it rose after that it never recovered to baseline levels. In other words, thedenominator (Cr) increased to a greater extent than the numerator (OHP) causing adecrease in the value of the ratio compared to the baseline measure.32Considering the difficulties in the measurement of OHP in this study, as well asthe results of Seaman and lanuzzo (35) and Horswill and colleagues (36), there is lack ofsupport for Abraham’s conclusion that DOMS is related to connective tissue damage inmuscle (6). However, differences in exercise protocols and differences in samplingprocedures in all of the studies are two major reasons why the results may differ and whyit is difficult to compare results. Further study is needed in this area, particularly studieswith similar protocols. Dietary restrictions are also an important consideration in OHPmeasurements. Subjects should abstain from foods high in collagen (meats, fish, poultryand foods containing gelatin) as urinary OHP is sensitive to dietary intake (37). Inaddition, the OHP urinalysis procedures outlined by Hughes and colleagues (24) requireda 24 hour urine collection. These criteria required a high degree of subject compliance,and non-compliance had the potential to contribute considerably to the variability. Fromthe results of this study, the fact that the standard deviation was high at the pre-testsuggests that diet and subject compliance may have been a factor contributing to thevariability of the OHP measure.There were significant correlations between the intensity of soreness and CPK,and between unpleasantness and CPK at the last two testing times (Table 4). The highestcorrelation (0.90) was between unpleasantness and CPK at 168 hours (7 days) after theshorter exercise stimulus. Even though the group responses had essentially returned tobaseline, those subjects with the highest CPK responses had the highest unpleasantnessscores.Rodenburg and colleagues (28) also have reported significant within-measurecorrelations and between-measure correlations for 27 subjects for DOMS and CPK at fivetime periods over 96 hours following eccentric exercise of the elbow flexors.Correlations among DOMS and CPK ranged from 0.36 to 0.58 from 48 to 96 hours post-33exercise. In this study signficant correlations were found between intensity of sorenessand CPK from 96 to 168 hours post-exercise (0.51- 0.76) and between unpleasantnessand CPK over the same time period (0.52 - 0.90) (Table 4). The highest correlations inthis study were at 168 hours post-exercise. Even though the group means were returningto baseline for these dependent variables, the high correlations suggest that there was arelationship between DOMS and CPK from four to seven days post-exercise.ConclusionsFrom the results of this study, there were no significant differences between thetwo exercise durations for any of the outcome measures. The primary difference in theprotocol between the two levels of exercise was in regard to duration, or number ofrepetitions, and although the shorter exercise duration (180 repetitions) was 60% that ofthe longer duration (300 repetitions) the outcome responses were essentially the same.This differs from the results of Tiidus and lanuzzo (18) but their exercise protocol wasconcentric and eccentric knee extension while the protocol in this study was eccentricknee extension only.The functional outcome measures (intensity of soreness, unpleasantness andeccentric torque) were all significant over time (p<0.001). The greatest perception of theintensity of soreness was between 24 and 48 hours for both exercise durations. Thegreatest perception of unpleasantness was at 48 hours post-exercise for both exercisedurations. The greatest loss of eccentric torque occurred at 24 hours post-exercise,34however, there were no significant correlations among eccentric torque and the otheroutcome measures.In this study, a sensory descriptor scale (DDS) that assessed more than onedimension of discomfort was utilized. Using the DDS, the subjects’ perception ofintensity of soreness as well as their perception of unpleasantness of soreness- theaffective or emotional response to discomfort - was examined. The results of this studysuggest that the affective domain was not a primary experience for the subjects, in thatthe highest unpleasantness response was much lower than that of the intensity ofsoreness.There were no significant differences between exercise durations or over time forthe biochemical outcome measures (CPK and OHP). The variability in the CPK responsemay have been due to the uneven numbers of males and females in this study, but thepattern of the responses was also similar to what has been reported previously. Thegreatest CPK response was 96 hours (four days) to 168 hours (seven days) post-exercise,which was later than the greatest perception of DOMS. The variability in the OH?response in this study may have been related to diet and the criteria for urine collection.Other investigators have reported a lack of strong relationships among theresponses of fatiguing eccentric exercise, a conclusion which supports the suggestion thatmore than one mechanism contributes to the characteristics of muscle injury aftereccentric exercise. Because there was only one strong correlation among the outcomemeasures over the two exercise durations and over time {CPK and unpleasantness 168hours (7 days) after the shorter exercise}, the results would suggest that, other than CPKand DOMS, the outcome measures examined in this study are not related.35References1. Armstrong RB. Mechanisms of exercise-induced delayed onset muscularsoreness: a brief review. Medicine and Science in Sports and Exercise 1984;16(6):529-538.2. Gracely RH, Kwilosz DM. The descriptor differential scale: applyingpsychophysical principles to clinical pain assessment. Pain 198835:279-288.3. Melzack R, Finch L. Objective pain measurement: a case for increased usage.Physiotherapy Canada 1981 34(6)(Nov!Dec).4. Whitaker OC, Warfield CA. The measurement of pain. Hospital Practice1988;(Feb): 15.5. Melzack R. Pain Measurement and Assessment. New York: Raven Press, 1983.6. Abraham WM. Factors in delayed muscle soreness. Medicine and Science inSports 1977;9: 11-20.7. Bobbert MF, Hollander AP, Huijing PA. Factors in delayed onset muscularsoreness of man. Medicine and Science in Sports and Exercise 1986;18(1):75-81.8. Dick RW, Cavanagh PR. An explanation of the upward drift in oxygen uptakeduring prolonged submaximal downhill running. Medicine and Science in Sports andExercise 1987;19(3):3 10-317.369. Schwane JA, Williams JS, Sloan JH. Effects of training on delayed musclesoreness and serum creatine kinase activity after running. Medicine and Science in Sportsand Exercise 1987;19(6):584-590.10. Clarkson PM, Nosaka K, Braun B. Muscle function after exercise-induced muscledamage and rapid adaptation. Medicine and Science in Sports and Exercise1992;24(5) :512-520.11. Schwane JA, Johnson SR. Vandennakker CB, Armstrong RB. Delayed-onsetmuscle soreness and plasma CPK and LDH activities after downhill running. Medicineand Science in Sports and Exercise 1983;15:51-56.12. Stauber WT, Clarkson PM, Fritz VK, Evans WJ. Extracellular matrix disruptionand pain after eccentric muscle action. Journal of Applied Physiology 1990;69(3):868-874.13. Smith L. Acute inflammation: The underlying mechanism in delayed onsetmuscle soreness? Medicine and Science in Sports and Exercise 1991;23:542-551.14. Ebbeling CB, Clarkson PM. Exercise-induced muscle damage and adaptation.Sports Medicine 1989;7:207-234.15. Stauber WT. Exercise and Sport Science Reviews.Baltimore: Williams andWilkins, 1989(vol 17):157-185. Eccentric action of muscles: physiology, injury, andadaptation.3716. Newham DJ, Mills KR, Quigley BM, Edwards RHT. Pain and fatigue afterconcentric and eccentric contractions. Clinical Science 1983;64:55-62.17. Fridën J, Sjostrom M, Ekblom B. Myofibnllar damage following intense eccentricexercise in man. International Journal of Sports Medicine 1983;4: 170- 176.18. Tiidus PM, lanuzzo DC. Effects of intensity and duration of muscular exercise ondelayed soreness and serum enzyme activities. 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Liquid chromatographic determination of 4-hydroxyproline in urine. Clinical Chemistry 1986;32(6): 1002-1004.25. Glass GV, Hopkins KD. Statistical Methods in Education and Psychology. (2 ed.)Boston: Allyn and Bacon, 198426. Appell HJ, Soares JM, Duarte JAR. Exercise, muscle damage and fatigue. SportsMedicine 1992;13(2): 108-115.27. Newham DJ, Jones DA, Clarkson PM. Repeated high force eccentric exercise:Effects on muscle pain and damage. Journal of Applied Physiology 1987;63: 1381-1386.28. Rodenburg JB, Bar PR, de Boer RW. Relations between muscle soreness andbiochemical and functional outcomes of eccentric exercise. Journal of AppliedPhysiology 1993 ;74(6):2976-2983.29. Faulkner JA, Brooks SV, Opiteck JA. Injury to skeletal muscle fibres duringcontractions: conditions of occurrence and prevention. Physical Therapy1993;73(12):91 1-921.30. Nosaka K, Clarkson PM, Apple FS. Time course of serum protein changes afterstrenuous exercise of the forearm flexors. Journal of Laboratory Clinical Medicine1992;119(2): 183- 188.31. Clarkson PM, Byrnes WC, Gillisson E, Harper E. Adaptation to exercise-inducedmuscle damage. Clinical Science 1987;73:383-386.3932. Hortobágyi T, Denahan T. Variability in creatine kinase: methodological,exercise, and clinically related factors. International Journal of Sports Medicine1989;10(2):69 - 80.33. Murguia MJ, Vailas A, Mandelbaum B, et al. Elevated plasma hydroxyproline Apossible risk factor associated with connective tissue injuries during overuse. AmericanJournal of Sports Medicine 1988;16(6):660-664.34. Stauber WT. Personal communication June, 1993.35. Seaman R, lanuzzo CD. Benefits of short-term muscular training in reducing theeffects of muscular over-exertion. European Journal of Applied Physiology1988;58(3):257-261.36. Horswill CA, Layman DK, Boileau RA, Williams BT, Massey BH. Excretion of3-methylhistidine and hydroxyproline following acute weight-training exercise.International Journal of Sports Medicine 1988;9(4):245-248.37. Elam RP, Hardin DH, Sutton RAL, Hagen L. Effects of arginine and ornithine onstrength, lean body mass and urinary hydroxyproline in adult males. The Journal ofSports Medicine and Physical Fitness 1989;29(1):52-56.40CHAPTER TWOTHE EFFECTS OF ECCENTRIC EXERCISE ON DELAYED MUSCLE SORENESS,MUSCLE STRENGTH AND FATIGUE, JOINT RANGE OF MOTION, ANDTHE PRESENCE OF LEUKOCYTES IN EXERCISED MUSCLEIntroductionA recent review by Smith (1) has revisited the hypothesis that the sensation of musclesoreness is associated with the acute inflammatory response. This hypothesis was initiallyproposed in the 1970’s but at that time was not supported by research findings. Smithreviewed these earlier reports and suggested that some discrepancies were apparent andthus some of the conclusions were not well-founded. Similarities between DOMS andacute inflammation include pain, swelling, and the loss of function as measured by musclestrength. Additionally, evidence of cellular infiltrates, such as macrophages, has beenreported, which have been noted to be present at the site of injury in animals from one tothree days (2, 3). Other indicators of inflammation such as the presence of fibroblasts (2)and increased lysosomal activity (4, 5) have also been found. Progression of the size ofthe lesion up to 48 hours has been observed (6), with signs of healing at 72 hours (2, 7).In a study of patients with inflammatory muscle disease, Yonker and colleagues (8)suggested that technetium-99m pyrophosphate (Tc-PYP) muscle scans are useful inestablishing sites of inflammation. Their results indicated that five of six patients withactive inflammatory muscle disease had positive scans, while nine patients without activedisease had negative scans. They concluded that the muscle scans separated the acuteinflammatory disease from inactive disease more consistently than muscle tenderness,increased CPK, muscle weakness or EMG.41Jones and colleagues (9) and Newham and colleagues (10) have reported on theutilization of Tc-PYP injections and scans after two different eccentric exercise regimes.They found an increased uptake of Tc-PYP into the exercised muscles and suggested thatthere may be a correlation with increased CPK but they did not assess the correlationstatistically. Oiily two or three subjects participated in each of the protocols. They alsoreported that little is known about the mechanism by which Tc-PYP is taken up intomuscle, although some have proposed an association between calcium accumulation andTc-PYP concentration (11). Thus, Tc-PYP may not be the best choice of an investigativetechnique to assess the underlying mechanism of muscle soreness, especially ifinflammatory mediators are associated with the soreness.Technetium-99m (Tc-99m) is a radioisotope that has certain advantages for clinicaluse - it has a short half-life of six hours, it has very good imaging characteristics, and thereis no beta radiation resulting in a lower radiation dose (12). Within the last two decades ithas also been used clinically as a radio label for white blood cells (WBC) to localizeinfections or abscess formation. As Evans and Cannon (13) and Smith and colleagues (14)have suggested that an increase in leukocytes in the first 24 hours after eccentric exercisewould be indicative of an acute phase inflammatory response, it follows that by labellingWBC, this may provide evidence of acute inflammation in muscle if there is an increasednumber of leukocytes within the muscle after eccentric exercise.Some investigators (4, 15) have reported upon decreased range of motion (ROM)after eccentric exercise and have suggested that this may be due to muscle shortening as aresult of calcium accumulation in damaged fibres. However, others (16, 17) haveconcluded that it might be edema within the perimuscular connective tissue that isresponsible for the restricted movement. Two studies (16, 18) have provided evidencethat the pressure from the increased fluid may also cause sensitization of the pain receptors42resulting in tenderness and muscle soreness. If edema is responsible for decreased ROMand sensitization of the pain receptors, then perception of DOMS and ROM should becorrelated.Faulkner and colleagues (19) have suggested that the initial loss of force, immediatelyafter the fatiguing eccentric exercise, is a function of both fatigue and injury to the muscletissue. They believe that recovery from fatigue occurs over the next three hours and iscomplete by 24 hours. Other investigators have studied the force/frequency characteristicsof eccentrically exercised muscles (20, 21). It has been found that there is a decrease inforce generation at low frequencies of stimulation and that this low frequency fatigue, orwhat may also be termed long-lasting fatigue, is more common with eccentric exercise thanconcentric. It has also been noted that this long-lasting fatigue is most commonimmediately after eccentric exercise (21). Jones and colleagues (22) have suggested thatmechanical damage to the muscle caused by the high forces of eccentric activity causes thelong-lasting fatigue.Power spectrum analysis has been widely employed to analyze muscle fatigue (23-25). It has been suggested that one of the factors that contributes to muscle fatigue is aslowing of the propagation velocity of the muscle fibre action potentials (24). Thisdecrease in the propagation velocity can contribute to a change of shape of the surfaceelectromyograph (EMG) signal during a prolonged isometric contraction (26). The changein EMG signal shape is measured as a shift in the frequency of the signal to a lowerfrequency (24). The power spectrum describes the relationship between signal amplitudeand signal frequency (24). The median frequency, which is a quantitative value, is anindex of the frequency shift (25) and thus, a measure of the slowing of the action potentialpropagation velocity.43In order to determine whether or not leukocytes were present in muscle followingeccentric exercise, a nuclear medicine technique was used to label WBC, and to monitortheir location over 24 hours. To determine whether or not other symptoms of acuteinflammation were present in the muscle, the perception of muscle soreness andunpleasantness was assessed, ROM of the knee joint was measured, and muscle strengthwas determined over 72 hours. In addition, to determine whether muscle fatigue waspresent after the exercise stimulus, assessment of muscle fatigue, utilizing power spectrumanalysis, was conducted over 72 hours following the eccentric exercise.PurposeIt was the purpose of this study to examine whether there was increased presence ofTc-99m WBC in the exercised muscle, compared to the contra-lateral non-exercised muscleover time. In addition, it was the purpose of this study to determine the time course andrelationships over time among DOMS, eccentric torque of the quadriceps muscles, musclefatigue of the quadriceps, range of motion (ROM) of the knee, and the presence of Tc-99mWBC in the exercised muscle.Research Hypotheses1. There will be greater presence of Tc-99m WBC in the exercised muscle compared to thecontra-lateral non-exercised muscle.2. The greatest perception of DOMS (measured as intensity of soreness andunpleasantness) will occur between 24 and 48 hours following the exercisestimulus, at the same time as the greatest presence of Tc-99m WBC and the greatestloss of ROM.3. The greatest decline in eccentric torque will occur in the first 24 hours following theexercise stimulus, at the same time as the onset of muscle fatigue, but before the44greatest perception of DOMS. Muscle fatigue will be measured as the medianfrequency of the power spectrum.Methods and proceduresSubjectsTwelve subjects volunteered for this study - 11 females and one male. The data fromone female subject was not included because one nuclear medicine scan time was missedand other test times became different from the protocol of this study. It was decided toanalyze only the data from the female subjects so that the subject group was morehomogeneous. Therefore, ten female subjects between the ages of 20 and 33 yearsparticipated in this study. Individuals were excluded from the study if they were pregnant,missed their last menstruation or were breast-feeding, or if they were engaged inrecreational exercise of more than six hours/week or involved in competitive sport, or ifthey had cardiovascular, neurological or musculoskeletal conditions that may havecompromised their ability to perform the testing procedures or which may have posed ahazard to the individual as identified by their physician (i.e.. - cardiac disease, a chronicneurological disease, degenerative knee joint disease, a recent soft tissue injury of the lowerextremity). Approval for this study was granted by the Clinical Screening Committee forResearch Involving Human Subjects at the University of British Columbia.ProtocolFollowing informed consent, subjects completed baseline measures of the DescriptorDifferential Scale (DDS) describing DOMS, eccentric torque of the right quadriceps, apower spectrum analysis of the right quadriceps, and range of motion (ROM) of the rightknee. Fifty milliliters (ml) of blood were taken by vempuncture. The WBC were sepamted45and labelled with Tc-99m HMPAO {(RR,SS)-4,8 diaza-3,6,6,9-tetramethyl undecane2,10-dione bisoxime} (See Appendix Four). The labelled WBC were re-introducedintravenously to the subjects immediately prior to the exercise stimulus. Following theexercise stimulus (300 repetitions), the DDS questionnaire, eccentric torque, powerspectrum analysis and ROM were repeated at two hours, four hours, 20 hours, 24 hours,48 hours and 72 hours. An extra test time for eccentric torque occurred at 0 hour at the endof the exercise stimulus. Bilateral scans of the quadriceps muscles (anterior and lateral)were taken at two hours, four hours, 20 hours and 24 hours after the exercise stimulus.Due to the decay of Tc-99m over 24 hours the data collection for Tc-99m WBC waslimited to 24 hours, while data collection for the other dependent variables continued for 72hours. As well, there was no pre-test for the presence of Tc-99m WBC but rather theexercise leg was compared to the non-exercise leg. This reduced the number of scan times.MethodsDescriptor Differential ScaleThe DDS was measured as described in Chapter One.Eccentric/Concentric Torque of the Quadriceps MusclesEccentric torque was measured the same as in Chapter One, except that data were alsocollected during the last set of the exercise stimulus (0 hour). In this case, repetitions twoto four were analyzed as the average eccentric torque of the knee extensors.46Power Spectrum AnalysisThe subjects were seated on the KinCom as described for the Eccentric/ConcentricTorque of the Quadriceps Muscles. The knee was positioned at 90 degrees of kneeflexion, the center of rotation of the KinCom was positioned opposite the knee joint lineand the lever arm was adjusted so that the resistance pad was placed on the distal aspectof the tibia as stated previously.The skin was cleaned with an alcohol swab and surface EMG electrodes (MediTrace silver/silver chloride, circular, 1 cm radius) were placed over the motor points ofthe vastus lateralis (VL), rectus femoris (RF), and vastus medialis (VM) musclesaccording to Delagi and colleagues (27). The interelectrode spacing was 2.5 cm. Aground electrode was placed over the wrist. Electrodes were placed on the skin withelectromedical gel between the skin and the electrodes. Subjects maintained a maximumisometric contraction for 60 seconds. A ten-second submaximal isometric contractionpreceded the one minute fatiguing contraction as a warm-up.The amplifier (custom-made) gain was set at 5000. The input impedance was 10megohms and the common mode rejection ratio (CMRR) greater than 100 dB. The rawsignal was amplified and then passed through a band pass filter with a frequency range of28-500 Hz. The force and EMG signals were simultaneously collected by the computer forspectral analysis. This analysis entailed partitioning the EMG data into overlapping four-second segments with each successive segment starting two seconds later than the previoussegment. Spectral estimates of each segment were then calculated. The median frequencyserved as the measure by which muscle fatigue was calculated. The median frequency ofeach four-second segment was plotted against time and a linear regression line was fitted tothe 28 data points. The slope of the plot was considered a quantitative measure of muscle47fatigue. The steeper the negative slope of the plot, the greater the muscle fatigue because asmuscle fatigues over time the spectral frequency shifts to a lower frequency. (SeeAppendix Three for further description of the power spectrum analysis).Range of MotionEach subject was positioned supine on a plinth. The centre of the goniometer waspositioned opposite the lateral aspect of the right knee joint line. Active range of motion(ROM) of the knee was measured with a plastic goniometer from full active extension tofull active flexion with the hip also fully flexed. ROM represented the painfree ROM thatthe subject could actively attain. The measured ROM did not differentiate between lack offull extension and/or loss of full flexion. A previous study by Rothstein and colleagues(28) reported the intra-tester reliability of goniometric measures of knee flexion andextension of 12 patients to range between 0.91 and 0.99 with a plastic goniometer. In theirstudy using the means of multiple measurements only improved the correlations slightly.The Exercise StimulusThe exercise stimulus was the same as outlined in Chapter One, except that in thisstudy the exercise stimulus required completion of 300 maximal eccentric contractionsonly. The subjects completed 30 sets of 10 repetitions with each set beginning everyminute for 30 minutes. This allowed a 20 second rest between each set.Radio-isotope Investigation of Acute InflammationThe WBC were separated from the blood sample and labelled with Tc-99m HMPAOaccording to procedures in the Nuclear Medicine Department, Vancouver Hospital48(Appendix Four). Immediately before the exercise stimulus the labelled WBC were reinjected intravenously into the subjects. Following the exercise stimulus, bilateral scans ofthe quadriceps muscles (anterior and lateral views) were taken at two, four, 20 and 24hours. As the half-life of Tc-99m is six hours and more than 90% of it has decayed by 24hours (12), the scan times were limited to within 24 hours. After the series of four scanswas completed, computer analysis of regions of interest (ROl) was used to determine thecount/pixel of gamma radiation. Four areas of the quadriceps muscle were chosen as theROT - the antero-distal aspect from the lateral view, the anterior aspect from the lateralview, the medial aspect from the anterior view and the lateral aspect from the anterior view(Figure 4).The analyses of the ROT were undertaken on three separate occasions by two scorers(observers). The ROT were carefully drawn to avoid the femur and the femoral circulation.The mean count/pixel of the three analyses were then calculated as well as the standarddeviation (SD). The coefficient of variation (CV) was calculated as the measure of inter-observer variability (Appendix Four). Next, the background, or noise, of the ROT of theexercise leg was subtracted. Tt is common in clinical practice to use the contra-lateral sideof an organ or segment of the body to represent the background, and in this case therespective ROT of the non-exercise leg was subtracted from the ROT of the exercise leg. Ttis also necessary to correct for the physical decay of the Tc-99m and this was done using adecay table to correct the count/pixel at each scan time (Appendix Four). Finally, asdosages of Tc-99m HMPAO varied between subjects, the counts/pixel were normalized tothe peak response of each subject and presented as a percentage so that responses could becompared between subjects who had different baselines (i.e.. - different dosages of Tc-99mHMPAO).49Figure 4. Regions of Interest for Radio-Isotope Scanning. The anterior view of boththighs is shown on the left. In the middle is the lateral view of the right thigh(exercise leg). On the right is the lateral view of the left thigh (non-exercise leg).The four regions of interest are identified on the figure.(3 cD51Statistical analysisA one-way repeated measures multivariate analysis of variance (MANOVA) was usedon each of the dependent variables (DDS intensity of soreness score, DDS unpleasantnessscore, eccentric torque, slope of the median spectral frequency of VL and VM, and ROM)to test for significant differences over time. A one-way repeated measures MANOVA wasused to examine the differences in the presence of Tc-99m labelled WBC in the quadricepsmuscles over time and between legs. As the “constant”, which is one source of variation inthe MANOVA analysis, represents the average of the means over the four test times andtests whether or not the average of the means is different from zero, and in our datapreparation each ROl of the non-exercise leg was subtracted from the ROT of the exerciseleg, it was appropriate to use the “constant” to determine whether or not the ROT of theexercise leg was significantly different from zero and thus, significantly different from thenon-exercise leg. As in the analyses of the other dependent variables, the source ofvariation “time” was used to determine the significant differences among the four timeperiods over time.Post hoc analyses examined contrasts between test times for all dependent variablesand at each test time compared to the pre-test for DOMS, eccentric torque, muscle fatigueand ROM. Only those variables that were significant over time were examined as themultivariate F test is more robust than the univariate post hoc tests (29). Correlationanalyses (Pearson product moment correlation) were used to determine relationships amongDOMS, eccentric torque, muscle fatigue and ROM. The significance level was set atp<O.O5.52ResultsThere was a significantly greater presence of Tc-99m WBC in the four ROl of theexercised muscle compared to the contra-lateral non-exercised muscle (p<0.0O1) (Table 8).The greatest presence at two hours post-exercise was in the antero-distal ROT and thepresence remained at approximately the same level over 24 hours (Figure 5A), while thepresence of Tc-99m WBC gradually increased over 20 and 24 hours in the other ROT(Figure 5).All of the dependent variables changed significantly over time (p<0.05) except theslope of the median spectral frequency for VM and the presence of Tc-99m WBC in theantero-distal ROl (Tables 7, 8). Intensity of soreness and unpleasantness were highest at24 hours post-exercise (Figure 7). The greatest loss of ROM also occurred at 24 hourspost-exercise (Figure 8). Eccentric torque was lowest at 0 hour, recovered, and thendeclined again at 20 to 24 hours post-exercise (Figure 6). The slope of the medianfrequency of vastus lateralis declined at two hours post-exercise (Figure 9). The greatestpresence of Tc-99m WBC occurred at 20 hours post-exercise in the antero-distal ROT andthe lateral ROT (Figure 5A and D). The greatest presence of Tc-99m WBC in the anteriorROI and the medial ROT was at 24 hours post-exercise (Figure SB and C). The means andstandard deviations for each of the dependent variables over time are presented in Tables 5and 6.The contrasts between the pre-test and each subsequent testing time were allsignificantly different (p.<0.05) for intensity of soreness, unpleasantness and ROM (Table9, Figures 7, 8). Each testing time up to and including 24 hours was significantly differentfrom the pre-test for eccentric torque (Table 9, Figure 6). Between testing times, therewere four significant contrasts between time periods for eccentric torque indicating theTABLE5INDIRECTMEASURESOFEXERCISE-INDUCEDMUSCLEINJURYBEFOREANDAFTERECCENTRICEXERCISEPre-test0Hour2Hours4Hours20HoursIntensityofSoreness-5.87±5.66.44±6.906.88±6.508.94±5.73Unpleasantness-8.77±2.24-3.63±3.34-2.21+3.601.80±3.66Torque(N.m)106.90±29.2665.90±22.1286.00±32.0089.4±28.1275.70±28.74ROM*(degrees)139.50±4.97136.80±6.16137.0±5.87134.50±5.50VLMedFreq(slope)-0.25±0.12-0.29±0.14-0.28±0.13-0.22±0.12VMMedFreq(slope)-0.22±0.11-0.23±0.12-0.23±0.20-0.22±0.2024Hours48Hours72HoursIntensityofSoreness11.11±3.5410.26±4.804.94±8.42Unpleasantness2.11±4.080.69±6.26-2.60±5.47Torque(N.m)73.90±29.1193.70±39.7894.20±35.27ROM(degrees)133.00±5.87133.80±6.32137.80±5.01VLMedFreq(slope)-0.21tO.13-0.20±0.10-0.23±0.11VMMedFreq(slope)-0.20±0.25-0.18±0.14-0.24±0.19Dataaremeans±SDfor tenfemalesubjects.*=Range ofMotion,+=Vastus LateralisMedianFrequencyandVastus MedialisMedianFrequency01 (.3TABLE6THEPRESENCEOFWHITEBLOODCELLSINFOURREGIONSOFINTERESTINTHEEXERCISEDMUSCLEAFTERECCENTRICEXERCISERegions ofInterest2Hours4Hours20Hours24HoursAntero-distal77.55±21.2268.54±21.0388.19±25.6777.24±28.18Anterior52.01±32.0542.80±28.3168.71±33.0781.42±21.14Medial32.77±22.5441.01±32.1465.49±34.0790.76±16.77Lateral45.53±42.1940.68±44.8190.59±16.5082.76±15.70Dataaremeans±SDfortenfemalesubjects.Theregionsofinterest weremeasuredascounts/pixel.c-fl55TABLE7MANOVA TABLES FOR THE INDIRECT MEASURESSource (of Variation) df MS FIntensity of SorenessWithin Subjects 54 23.67Time 6 326.55 13.79*UnpleasantnessWithin Subjects 54 12.55Time 6 145.34 11.58*Eccentric TorqueWithin Subjects 63 225.54Time 7 1758.21 7.80*Range of MotionWithin Subjects 54 8.42Time 6 55.33 6.57*Vastus Lateralis Median FrequencyWithin Subjects 54 0.00Time 6 0.01 5.31*Vastus Medialis Median FrequencyWithin Subjects 54 0.01Time 6 0.00 0.37* p<0.0156TABLE8MANOVA TABLES FOR THE PRESENCE OF WHITE BLOOD CELLS IN FOURREGIONS OF INTERESTSource (of Variation) df MS FAntero-Distal Region of InterestWithin Subjects 9 578.18Constant 1 242625.79 419.64*Within Subjects 27 589.25Time 3 646.57 1.10Anterior Region of InterestWithin Subjects 9 763.55Constant 1 149993.91 196.44*Within Subjects 27 868.41Time 3 2960.73 3.41kMedial Region of InterestWithin Subjects 9 885.95Constant 1 132274.15 149.30*Within Subjects 27 698.71Time 3 6844.49 9.80*Lateral Region of InterestWithin Subjects 9 2075.06Constant 1 168432.38 81. 17*Within Subjects 27 744.10Time 3 6470.23 8.70** p<0.01+ p<0.0557TABLE9POST HOC CONTRASTS OF THE INDIRECT MEASURESSource Umvariate F pIntensity of Soreness df 1,9pre-2hr 17.96 0.002*pre-4hr 20.85 0.001*pre-2Ohr 27.12 0.001*pre - 24 hr 43.54 <.001 *pre-4Shr 37.64 <.001*pre-72hr 15.89 0.003*2hr -4 hr 0.25 0.6314hr-2Ohr 4.92 0.0542Ohr-24hr 2.21 0.17124hr-48hr 0.62 0.45048 hr -72 hr 4.76 0.057Unpleasantness df 1,9pre-2hr 19.94 0.002*pre-4hr 27.70 0.001*pre-2Ohr 69.48 <.001*pre-24hr 44.22 <.001*pre-48hr 16.50 0.003*pre-72hr 12.16 0.007*2hr-4hr 16.45 0.003*4hr-2Ohr 9.95 0.012*2Ohr-24hr 0.76 0.78924hr-48hr 1.64 0.2334Bhr-72hr 1.96 0.195Eccentric Torque df 1,9pre - 0 hr 45.49 <.001 *pre-2hr 6.70 0.029*58Source Umvariate F pEccentric Torque cont.pre-4hr 5.54 0.043*pre-2Ohr 17.52 0.002*pre - 24 hr 23.67 <.001 *pre -48 hr 1.72 0.222pre-72hr 2.23 0.169Ohr-2hr 6.30 0.033*2hr-4hr 0.81 0.3924hr-2Ohr 12.83 0.006*2Ohr-24hr 0.18 0.68524hr-48hr 16.11 0.003*48 hr -72 hr 0.02 0.884Range of Motion df 1,9pre-2hr 5.20 0.048*pre-4hr 9.00 0.015*pre-2Ohr 15.00 0.004*pre-24hr 37.10 <.001*pre-48hr 16.80 0.003*pre-72hr 5.19 0.049*2hr-4hr 0.07 0.8014 hr -20 hr 3.46 0.0962Ohr-24hr 1.98 0.19324 hr - 48 hr 0.32 0.58348hr-72hr 16.00 0.003*Vastus Lateralis Median Frequency df 1,9pre-2hr 3.17 0.109pre -4 hr 1.45 0.260pre-2Ohr 1.15 0.311pre-24hr 1.76 0.21759Source Umvariate F pVastus Lateralis Median Frequency cont.pre-48hr 2.32 0.162pre-72hr 0.64 0.4442hr-4hr 0.54 0.4824hr-20hr 13.46 0.005*2Ohr-24hr 1.22 0.29924hr-48hr .0.01 0.93748 hr-72 hr 1.86 0.206* significant differences between tests60Figure 5. The Presence of White Blood Cells in the Four Regions of Interest in theExercised Muscle. Values are means ± SD. The presence of white blood cells inthe exercised muscle was significantly greater than in the contra-lateral non-exercised muscle (p<O.OO1) in all four regions.A. Antero-Distal Region of Interest.B. Anterior Region of Interest. Significant differences over time p<O.O5.C. Medial Region of Interest. Significant differences over time p<O.OO1.D. Lateral Region of Interest. Significant differences over time p<O.OO1.* Significantly different this test to previous test p<O.O110152025TIME(Hours)510152025TIME(Hours)Cs)0 C-)Ui1—i-J z 0 aFigure5100A80 60 40 20 0S4.4.IIII0510152025TIME(Hours)II____F05100C80 100D80be U) I a60Li 0 UitJ400 a20 010152025TIME(Hours)100B80be I 0S062Figure 6. Characteristics of Eccentric Exercise: Eccentric Torque.Values are means L SD. There were significant differences over time p<O.OO1.* Significantly different from the pre-test p<O.05. + Significantly different fromthe pre-test p<O.O 1. * * Significantly different from the pre-test p<O.OO1.++ Significantly different this test to previous test p<O.05. Significantlydifferent this test to previous test p<O.O1.-1 m 0 C (JECCENTRICTORQUE(Nm)---r’J-ZD0r’).00000000(D1 (D 0 0 r’J—1r’JO•)U)64Figure 7. Characteristics of Eccentric Exercise: Muscle Soreness.Values are means ± SD. There were significant differences over time p<O.OO1.* Significantly different from the pre-test p.cO.O 1. + Significantly different fromthe pre-test p<O.OO1.A. Intensity of SorenessB. Unpleasantness. * * Significantly different this test to previous test p<O. 05.++ Significantly different this test to previous test p.<O.O1.MEANSCOREMEANSCOREo0oor————r—-—-rji..—..r..i__7-->-oI-o(1).....J-‘...II’—S——N.)--I5+SN.)Io+C)*1.N.)--—-+....-a.am---0a0CIC.1--,--U,aU,-K-,aa...-*co+.—II•I—•I—I1I1—IjII-.IIIS I..I-*1—.—IIF——IIIII.L_—0 0,66Figure 8. Characteristics of Eccentric Exercise: Range of Motion of the Knee.Values are means ± SD. There were significant differences over time p<O.OO1.* Significantly different from the pre-test p<O.05. + Significantly different fromthe pre-test p<O.O 1. * * Significantly different from the pre-test p<O.OO1.++ Significantly different this test to previous test p<O.O1.RANGEOFMOTION(degrees)---rJLuL&)-(‘-ILI,0U,0U,__.._.____._._l____[--.ODIIINi•----IINJ 0+N).---1-----Im 0 -‘N)+-________68Figure 9. Slopes of the Median Frequencies of Two of the Quadriceps Muscles.Values are means ± SD.A. Median Frequency of the Vastus Lateralis Muscle. Significant differences overtime p<O.OO1. * Significantly different this test to the previous test p<O.Ol.B. Median Frequency of the Vastus Medialis Muscle.SLOPEOFTHEMEDIANFREQUENCYSLOPEOFTHEMEDIANFREQUENCYIII000p-IU)PICx)PICOI\)Pp0-0(11Cnci,U,U,U,iU,U,Ci,CITCDI;I::*II70changes that were occurring in muscle strength over time (Table 9, Figure 6). Thesignificant contrasts for unpleasantness were between two and four hours and between fourand 20 hours post-exercise, as the perception of unpleasantness was increasing (Table 9,Figure 7B). For ROM, there was significant change between 48 and 72 hours post-exercise as ROM was increasing again (Table 9, Figure 8). The only significant contrastfor VL median frequency was between four and 20 hours as the slope of the medianfrequency was increasing (Table 9, Figure 9A). There was only one significant contrastfor the lateral ROl between four and 20 hours post-exercise as the presence of Tc-99mWBC was increasing (Table 10, Figure 5D).Table 11 is a summary of the correlations among the dependent variables, presentedas correlation matrices. Although it was originally proposed to correlate the otherdependent variables with the presence of Tc-99m WBC, these correlations have not beenreported because the Tc-99m WBC data was normalized. Upon plotting some of the data itwas noted that the normalized values of 100% created a “ceiling effect” which did notrepresent the range of values for each subject. However, the non-normalized data did notaccount for the differences between the dosages of Tc-99m HMPAO.ROM and unpleasantness were significantly correlated at 20 hours post-exercise(r=-0.77). Other significant correlations between eccentric torque and vastus lateralismedian frequency (r= -0.65) and vastus medialis median frequency (r= -0.68) were foundat two hours post-exercise, and again at 20 hours for vastus medialis median frequency(r= -0.64). Significant correlations were found between intensity of soreness andunpleasantness at the pre-test (r=0.73) and at 72 hours (r=0.85) following the exercisestimulus (Table 11).71TABLE 10THE PRESENCE OF WHITE BLOOD CELLS IN THE FOUR REGIONS OFINTEREST:POST HOC CONTRASTSSource Umvariate F pAnterior Region of Interest df 1,92hr-4hr 1.09 0.3234 hr -20 hr 4.22 0.0702Ohr-24hr 1.11 0.320Medial Region of Interest df 1,92hr-4hr 1.07 0.3284hr-2Ohr 3.58 0.0912Ohr-24hr 2.97 0.119Lateral Region of Interest df 1,92hr-4hr 0.24 0.6374hr-2Ohr 14.74 0.004*20 hr 24 hr 0.77 0.402* significant differences between tests72TABLE 11RELATIONSHIPS AMONG MUSCLE SORENESS, MUSCLE STRENGTH ANDFATIGUE, AND RANGE OF MOTION AT EACH TEST TIMECORRELATION MATRICESTest Times (hours)Pre-test 2 4 20 24 48 72Intensity of SorenessTest Times (hours)UnpleasantnessPre-test 0.73*2 0.524 0.5020 0.1824 -0.1848 0.5972 0.85*Eccentric TorquePre-test 0.542 0.514 0.2520 0.0124 -0.4148 -0.5372 0.04Median Frequency of Vastus LateralisPre-test -0.352 -0.444 O.71*20 -0.4424 -0.3648 -0.2372 -0.56Median Frequency of Vastus MedialisPre-test 0.112 -0.174 -0.4620 -0.2424 -0.2848 -0.3572 -0.47Test Times (hours)Pre-test 2 4 20 24 48 72Intensity of Soreness cont.Test Times (hours)Range of MotionPre-test -0.592 0.044 0.1120 -0.2124 0.38‘48 -0.3372 0.02UnpleasantnessEccentric TorquePre-test 0.552 0.194 0.1320 0.2224 0.3848 -0.3072 -0.20Median Frequency of Vastus LateralisPre-test -0.112 -0.274 -0.4520 -0.2424 0.0748 0.0272 -0.48Median Frequency of Vastus MedialisPre-test 0.162 0.074 0.0420 -0.2524 0.3048 -0.0372 -0.30Range of MotionPre-test -0.552 -0.414 -0.0920 O.77*24 -0.3048 -0.4772-0.287374Test Times (hours)Pre-test 2 4 20 24 48 72Eccentric TorqueTest Times (hours)Median Frequency of Vastus LateralisPre-test -0.382 .0.65*4 -0.2120 -0.4824 -0.00148 -0.2572 -0.26Median Frequency of Vastus MedialisPre-test -0.052 .0.68*4 -0.2220 .0.64*24-0.3248-0.0372-0.31Range of MotionPre-test -0.292 -0.074-0.0820 -0.0424 0.0248-0.0172 0.002Range of MotionMedian Frequency of Vastus LateralisPre-test 0.242 0.074 0.0720 0.2424-0.0648 0.1472-0.02Median Frequency of Vastus MedialisPre-test -0.222 0.364 0.1520 0.1124 0.1848-0.2372 0.03* p<0.575DiscussionTogether with indirect measures of muscle damage, a technique to measure themuscle damage directly should also be employed (19). In this study the presence of Tc99m WBC in the exercised muscle compared to the contra-lateral non-exercised muscle wasmonitored over 24 hours following eccentric exercise. In all of the ROl the presence of Tc99m WBC in the exercised quadriceps muscles was significantly greater (p<0.0O1) than inthe contra-lateral non-exercised muscle (Table 8). The presence of Tc-99m WBC alsoincreased up to 20 and 24 hours post-exercise, except for the antero-distal aspect whichremained high from two to 24 hours post-exercise (Figure 5). In addition, there was asignificant increase in the presence of Tc-99m WBC between two hours and 20 hours post-exercise for the lateral aspect. However, because the presence of Tc-99m WBC was notmonitored beyond 24 hours, it would be premature to conclude that the greatest presence ofWBC in the muscle occurred at 20 and 24 hours following eccentric exercise.The significance of these findings are two-fold. First, the results show thatinflammatory cells were present in the exercised muscle of humans in the 24 hoursfollowing eccentric exercise, similar to what occurs in animals (2). In this study the Tc99m WBC were found to be in significantly greater numbers in the exercised quadricepsmuscles compared to the contra-lateral non-exercised muscles over the first 24 hours postexercise.Secondly, the greatest presence of Tc-99m labelled WBC immediately post-exercisewas into the antero-distal ROl of the exercised muscle (Figure 5A). However, it wasevident that the WBC were present throughout the muscle in increasing numbers over 24hours (Figure 5). This nuclear medicine technique allowed a quantitative evaluation of the76widespread distribution of the exercise-induced muscle damage, as reflected by thepresence of inflammatory cells in the exercised muscle, compared to the smaller sample thatwould have been evaluated from microscopic examination of human muscle biopsies.Although the WBC presence was greatest in the antero-distal ROl of the exercised muscletwo hours post-exercise and this continued to be the most visually obvious site of increasedTc-99m WBC over 24 hours, the increased WBC in the other ROT also suggests evidenceof inflammation throughout the entire quadriceps muscle.Due to the normalized data, it was not possible to correlate the presence of Tc-99mWBC with the other dependent variables. However, during the data collection subjectswere asked to subjectively report the location of their muscle soreness by indicating on abody diagram where they felt the soreness. At two hours post-exercise nine out of the tensubjects reported their soreness in a distal location on the right anterior thigh. By 24 - 48hours post-exercise all of the subjects reported soreness proximally through the quadricepsmuscles. These reports are similar to the timing of the increasing presence of Tc-99mWBC in the four ROT. Although this is anecdotal information, Newham and colleagues(21) have also reported tenderness beginning medially, laterally and distally and thenbecoming more diffuse throughout the quadriceps muscles by 24 - 48 hours after theexercise. These findings suggest further study. If it is possible to keep the dosages of theradio-isotope constant, then it would be possible to compare the measure of acuteinflammation to the other dependent variables, such as intensity of soreness and eccentrictorque, following eccentric exercise.As indicated in the protocol, additional eccentric torque data were collected at the endof the exercise stimulus (0 hour) as well as at the other time periods of the protocol. Bycollecting data between the end of the exercise stimulus and 24 hours after, it can be seenthat there was some recovery of torque after the first decline at 0 hour but then it declined77again at 20 and 24 hours post-exercise (Figure 6). Faulkner and colleagues (19) havereported this pattern of response in mice but to date this has not been reported in humans.Most other investigators have reported the greatest decline in force in humans immediatelyfollowing the exercise with recovery at 24 hours and onwards (4, 22, 30), but in theseprevious studies, data were not collected between one hour and 24 hours post-exercise.The second decline in eccentric torque in this study occurred from 20 - 24 hours(Figure 6). The peak intensity of soreness and unpleasantness occurred at the same time.This suggests that the intensity of soreness and unpleasantness may be related to asecondary response to the original injury to the muscle but there were no significantcorrelations between eccentric torque and either intensity of soreness or unpleasantness(Table 11). The different response patterns during the first 24 hours between eccentrictorque and either intensity of soreness or unpleasantness may explain why no studies havefound a definitive relationship between DOMS and muscle strength.The subjects’ perception of intensity of soreness and unpleasantness both peaked at24 hours following the exercise stimulus (Figure 7). Although every time period wassignificantly different from the pre-test, there was little difference between successiveresponses from two to 72 hours for intensity of soreness and from 20 to 72 hours forunpleasantness. Reports in the literature have stated that the greatest sensation of sorenessoccurs between 24 and 48 hours in the quadriceps muscles (21) and between 48 and 72hours in the elbow flexors (30, 31). In this study three additional time periods of data werecollected between the pre-test and the usual reporting time of 24 hours. The responses attwo, four and 20 hours after the eccentric exercise confirmed that the intensity of sorenessand unpleasantness progressively increase for up to 24 hours. Previous testing (ChapterOne) revealed that both intensity of soreness and unpleasantness responses had returned to78baseline levels by seven days but because the protocol ended at 72 hours in this studyneither had returned to the pre-test levels.Another classic symptom of acute inflammation is the formation of edema or swelling(1). Based on the hypothesis that swelling within the muscle tissue will result in decreasedROM and increased discomfort within the first 48 hours of the eccentric exercise (16), theresults of this study indicate significant loss of ROM up to 24 hours post-exercise (Figure8) a pattern similar to the inverse response of the intensity and unpleasantness of soreness(Figure 7).Although the changes in ROM were not significantly correlated with intensity ofsoreness, there was a significant inverse correlation (r=-O.77,r2=O.59) between ROM andunpleasantness at 20 hours (Table 11). The higher the unpleasantness score for a subject,the less the ROM at the knee. The single significant correlation should be interpretedcautiously but by assessing more than one dimension of the discomfort of muscle soreness,additional information regarding the onset of muscle soreness may be gathered.As the pain receptors are within the connective tissue surrounding the muscle andgroup IV sensory fibres terminate as free nerve endings in this same region (5), therelationship between decreased ROM and unpleasantness lends support to the mechanicaleffect of swelling within the connective tissue causing increased pressure and thusdiscomfort. The lack of a relationship between eccentric torque and either intensity ofsoreness or unpleasantness (Table 11) provides some evidence that the pain receptors in theconnective tissue appear to be reflecting the muscle injury differently than the functionalresponse of the muscle itself. Stauber (5) believes that the pain receptors may beresponding to swelling of the endomysium and perimysium. Faulkner and colleagues (19)suggest that the changes in muscle strength are reflecting the damage to the myofiber.79Finally, in order to determine whether or not the initial loss of strength immediatelyafter the strenuous eccentric exercise was due to muscle fatigue, a power spectrum analysisof the superficial quadriceps muscles was conducted. The results for the rectus femorismuscle indicated that the sitting position, with rectus femoris acting over two joints, wasnot the best position for assessing the fatigue of this muscle. Further explanation of theresults for rectus femons are presented in Appendix Three.The slope of the median frequency for vastus lateralis decreased at two hours andthen increased up to 20 hours post-exercise but the median frequency for vastus medialisdid not change significantly over time (Table 7). The lack of significance for vastusmedialis was most likely due to the small differences between time periods and thevariability in the responses (Figure 9).Roy (24) has commented that there is renewed interest in EMG spectralmeasurements for evaluating muscle fatigue due to the technical advances of digital systemsand computer memory and processing speed. He reports that it has been used inergonomics to assess muscle fatigue in occupational tasks and to assess muscle impairmentin low back pain and neuromuscular disorders. However, he still considers it in thedevelopmental stage. In this study there was a statistically significant difference over timein VL only. The decline in the slope of the median frequency of vastus lateralis at twohours post-exercise suggested that the greatest fatigue occurred in the muscle at this timebut the difference between the pre-test and two hours post-exercise was not statisticallysignificant (Figure 9). (See Appendix Three for further discussion.)Muscle fatigue has been reported by others (21, 22) immediately following eccentricexercise and the pattern of response of VL in this study is similar to those reports.80Newham and colleagues (21) reported recovery from fatigue by 24 hours while Jones andcolleagues (22) reported three to four days for recovery. In this study VL appeared to haverecovered from fatigue by 20 hours post-exercise.The significant negative correlations between eccentric torque and the medianfrequency of VL and the median frequency of VM at two hours and 20 hours post-exercise(Table 11) indicated that those subjects with the greatest eccentric torque were those whohad the greatest decline in the slopes of the median frequencies of VL and/or VM, and viceversa. The greatest decline in the slope of the median frequency was equivalent to thegreatest fatigue of the muscle. The relationship that was expected was a positivecorrelation. In order to understand these relationships, first the data was plotted andsecondly, the slopes of the median frequencies of VL and VM were compared to thedeclining force of the quadriceps muscles during the 60 second isometric contraction of thepower spectrum analysis.From the plotted data it was obvious that an outlier existed. Figure 10 is an exampleof the plot of the correlation between eccentric torque and the slope of the median frequencyof VL at two hours post-exercise. This same outlier was present in the three significantcorrelations between eccentric torque and the slopes of the median frequencies. When therelationships were examined without the outlier none of the correlations were significant -all three were equal to or less than r= -0.30.Further examination of the slopes of the median frequencies of VL and VM and theslope of the declining force of the quadriceps muscles during the 60 second isometriccontraction revealed a similar relationship as the one with eccentric torque. A single outlier(the same subject) transformed the lack of a relationship in the data into a significant linear81Figure 10. The Relationship Between Eccentric Torque and Vastus Lateralis MedianFrequency at Two Hours Post-Exercise. TOR2 = eccentric torque, measured inNewton-meters. VL2 = the slope of the median frequency of the vastus lateralismuscle. The responses for each of the ten subjects are illustrated.82Figure 10150 .IT0R2I .50I0 I I-0.6 -0.5 -0.4 -0.3 -0.2 -0.1VLZ83relationship. Without this outlier there was no pattern in the data from the other ninesubjects.These two tests of muscle activity were different in that eccentric torque measured theforce generating capability of the muscle during four maximal repetitions through 60degrees of ROM at a velocity of 30 degrees/second. The power spectrum analysis assessedmuscle fatigue, the progressive impairment of the force-generating capacity of the muscle(24), which involved a 60 second isometric contraction at 90 degrees of knee flexion.The lack of a correlation between eccentric torque and the power spectrum analysisindicates that other central or peripheral factors may be contributing to the initial loss ofstrength in the muscle. Peripheral factors may include damage to the sarcomere (19), orloss of intracellular Ca homeostasis (32), or damage to the connective tissue (16), butthe pattern of response of VL suggests that muscle fatigue should not be eliminated as oneof the factors contributing to the decline in eccentric torque.Another significant correlation between the slope of the median frequency of VLand intensity of soreness (r=-0.71) was found at four hours post-exercise (Table 11). Itappeared that those subjects with the highest intensity of soreness score had the mostfatigue in VL. However, once again, a single outlier was affecting the correlation. Theoutlier in this correlation represented a different subject but without this data point the linearrelationship was not significant (r=-0.46). Further study of the relationships among thepower spectrum analysis and the other dependent variables might benefit from largersubject numbers to either confirm or refute these results.84ConclusionsThe significant differences in the presence of Tc-99m WBC between the sore,exercised muscle and the contra-lateral non-exercised muscle shows that leukocytes werepresent in the muscle in the first 24 hours following eccentric exercise. The substantialpresence of Tc-99m WBC in the antero-distal aspect of the thigh at two hours post-exercise, which remained at a high level for 24 hours, suggests that there may be greaterdamage to the muscle in this region due to increased stess at the muscle/tendon junction.All of the dependent variables were significant over time except VM median spectralfrequency and the presence of Tc-99m WBC in the antero-distal ROT. Intensity of sorenessand unpleasantness were highest at 24 hours post-exercise while the loss of ROM wasgreatest at 24 hours post-exercise. The presence of Tc-99m WBC in the anterior, medialand lateral ROI increased up to 20 and 24 hours post-exercise but data collection did notproceed beyond 24 hours. The median frequency of vastus lateralis declined at two hourspost-exercise.In this study data were collected between the end of the exercise stimulus and 24hours, at two, four and 20 hours post-exercise. By doing so a biphasic pattern in theresponse of eccentric torque was illustrated in humans that has only been reportedpreviously in animal studies. Eccentric torque declined at 0 hour, recovered, and thendeclined again at 20 to 24 hours post-exercise. This pattern of response, which wasdifferent from intensity of soreness, unpleasantness, ROM and fatigue in this study,provides some explanation as to why measures of muscle strength have not correlated withother outcome measures of exercise-induced muscle damage in the muscle injury literature.The biphasic response of eccentric torque also supports the suggestion that there is morethan one mechanism underlying exercise-induced muscle injury.85The only significant correlations in this study that had a linear relationship and wererepresentative of the data, were between ROM and unpleasantness at 20 hours post-exercise, and between intensity of soreness and unpleasantness at the pre-test and 72 hourspost-exercise. The lack of a relationship between eccentric torque and either VL medianfrequency or VM median frequency in nine out of ten subjects, suggests that other factors,in addition to muscle fatigue, may be contributing to the decline in eccentric torque after theexercise stimulus.86References1. Smith L. Acute inflammation: The underlying mechanism in delayed onset musclesoreness? Medicine and Science in Sports and Exercise 1991;23:542-551.2. Armstrong RB, Ogilvie RW, Schwane JA. Eccentric exercise-induced injury to ratskeletal muscle. Journal of Applied Physiology 1983; 54:80-93.3. Zerba E, Komorowski TE, Faulkner JA. Free radical injury to skeletal muscles ofyoung, adult and old mice. American Journal of Physiology 1990;258: c429- c435.4. Ebbeling CB, Clarkson PM. Exercise-induced muscle damage and adaptation.Sports Medicine 1989;7: 207-234.5. Stauber WT. Exercise and Sport Science Reviews.Baltimore: Williams andWilkins, 1989(vol 17): 157-185. Eccentric action of muscles: physiology, injury, andadaptation.6. Newham DJ, Jones DA, Ghosh G, Aurora P. Muscle fatigue and pain aftereccentric contractions at long and short length. Clinical Science 1988:74:553-557.7. Fridén J, Sjostrom M, Ekblom B. Myofibrillar damage following intense eccentricexercise in man. International Journal of Sports Medicine 1983;4: 170- 176.8. Yonker RA, Webster EM, Edwards NL, et al. Technetium pyrophosphate musclescans in inflammatory muscle disease. British Journal of Rheumatology 1987;26:267-269.879. Jones DA, Newham DJ, Round JM, Toifree SEJ. Experimental human muscledamage: morphological changes in relation to other indices of damage. Journal ofPhysiology 1986;375:435-44&10. Newham DJ, Jones DA, Toifree SE, Edwards RH. Skeletal muscle damage: astudy of isotope uptake, enzyme efflux and pain after stepping. European Journal ofApplied Physiology 1986;55(1): 106-112.11. Willerson if, Parkey RW, Bonte FJ, Lewis SE, Corbett J, Buja LM.Pathophysiologic considerations and clinicopathological correlates of technetium-99mstamious pyrophosphate myocardial scintigraphy. Seminars in Nuclear Medicine1980;10(1):54-69.12. Kowalsky RJ, Perry JR. Radiopharmaceuticals in Nuclear MedicinePractice.Norwalk, CT: Appleton and Lange, 198713. Evans WJ, Cannon JO. Exercise and Sports Science Reviews.Baltimore: Williamsand Wilkins, 1991(vol. 19):99-125. The metabolic effects of exercise-induced muscledamage.14. Smith LL, McCammon M, Smith S, Chamness M, Israil RG, O’Brien KR Whiteblood cell response to uphill walking and downhill jogging at similar metabolic loads.European Journal of Applied Physiology 1989;58:833-837.15. Clarkson PM, Tremblay I. Exercise-induced muscle damage, repair, and adaptationin humans. Journal of Applied Physiology 1988;65: 1-6.8816. Jones DA, Newham DJ, Clarkson PM. Skeletal muscle stiffness and painfollowing eccentric exercise of the elbow flexors. Pain 198730:233-242.17. Howell JN, Chila AG, Ford G, David D, Gates T. An electromyographic study ofelbow motion during postexercise muscle soreness. Journal of Applied Physiology1985;58(5): 1713-1718.18. Fridén J, Sfakianos PN, Hargens AR, Akeson WH. Residual muscular swellingafter repetitive eccentric contractions. Journal of Orthopaedic Research 1988;6:493-498.19. Faulkner JA, Brooks SV, Opiteck JA. Injury to skeletal muscle fibres duringcontractions: conditions of occurrence and prevention. Physical Therapy 1993 ;73( 12): 911-921.20. Davies CTM, White MJ. Muscle weakness following eccentric work in man.Pflugers Archiv 1981 ;392: 168- 171.21. Newham DJ, Mills KR, Quigley BM, Edwards RHT. Pain and fatigue afterconcentric and eccentric contractions. Clinical Science 1983 ;64: 55-62.22. Jones DA, Newham DJ, Torgan C. Mechanical influences on long-lasting humanmuscle fatigue and delayed-onset pain. Journal of Physiology 1989;412:415-427.23. Frascarelli M, Rocchi L, Feola I. EMG computerized analysis of localized fatigue induchenne muscular dystrophy. Muscle and Nerve 1988;1 1:757-76 1.8924. Roy SH. Combined use of surface electromyography and P3 1-NMR spectroscopyfor the study of muscle disorders. Physical Therapy 1993 ;73(12):892-901.25. Tarkka TM. Power spectrum of electromyography in arm and leg muscles duringisometric contractions and fatigue. Journal of Sports Medicine 1984;24: 189-194.26. Zwarts MJ, Van Weerden TW, Haenen HTM. Relationship between averagemuscle fibre conduction velocity and emg power spectra during isometric contraction,recovery and applied ischemia. European Journal of Applied Physiology 1987;56:212-216.27. Delagi EF, Perotto A, lazetti J, Morrison D. Anatomic Guide for theElectromyographer.Springfield, IL: C. Thomas, 197528. Rothstein JM, Miller PJ, Roettger RF. Goniometnc reliability in a clinical setting.Physical Therapy 1983;63(1O): 161 1-1615.29. Glass GV, Hopkins KD. Statistical Methods in Education and Psychology. (2 ed.)Boston: Allyn and Bacon, 198430. Clarkson PM, Nosaka K, Braun B. Muscle function after exercise-induced muscledamage and rapid adaptation. Medicine and Science in Sports and Exercise1992;24(5):5 12-520.31. Rodenburg JB, Bar PR, de Boer RW. Relations between muscle soreness andbiochemical and functional outcomes of eccentric exercise. Journal of Applied Physiology1993 ;74(6): 2976-2983.32. Armstrong RB. Initial events in exercise-induced muscular injury. Medicine andScience in Sports and Exercise 1990;22(4):429-435.9091CHAPTER THREECONCLUDING REMARKSCollecting data after the exercise stimulus and before 24 hours revealed the biphasicresponse of eccentric torque following eccentric exercise which has not been reportedbefore in humans. Eccentric torque decreased at 0 hour, recovered, and then decreasedagain at 20 to 24 hours post-exercise. This response may explain why eccentric torquedoes not correlate with other outcome measures. It would also support the hypothesis thatmore than one mechanism underlies exercise-induced injury in muscle.Two of the proposed mechanisms associated with loss of muscle strength followingeccentric exercise are muscle fatigue immediately after the exercise stimulus and abiochemical response to the phagocytic activity at 24 hours post-exercise (1). The resultsof this study indicate that fatigue, measured as slowing of the action potential conductionvelocity, may be related to the loss of strength, in that the slope of the median frequency ofvastus lateralis (VL) declined at two hours post-exercise but it was not significantlycorrelated with eccentric torque in nine out of ten subjects. Muscle fatigue may be moreimportant to the loss of muscle strength earlier than two hours in the post-exercise period.The difference between the means of the median frequency of VL at the pre-test and twohours post-exercise in the study outlined in Chapter Two was small (0.04), while thedifference between the means at the pre-test and 0 hour in the pilot study outlined inAppendix Three was larger (0.13 1). This is an area of further study, which would alsobenefit from a correlation analysis of torque and fatigue in the first few hours post-exercise.Factors other than fatigue may also contribute to the loss of force immediately afterthe eccentric exercise. Damage to the sarcomere may physically limit the ability of the92muscle to produce force (1) or loss of calcium homeostasis may contribute further to theinitial injury (2) and it may affect the functioning of the contractile unit (3).In nine out of ten subjects who participated in the study outlined in Chapter Two,the region of interest (ROT) with the greatest presence of technetium-99m white blood cells(Tc-99m WBC) at two hours post-exercise was the antero-distal ROT. It appears that thisROT represented the muscle/tendon junction of the quadriceps muscle and that the leukocyteresponse to the injury was earlier than in the other ROT. The presence of Tc-99m WBC inthe other ROT gradually increased up to 20 and 24 hours post-exercise while the presenceof Tc-99m WBC in the antero-distal ROI was high at two hours post-exercise and remainedhigh over the 24 hours. These results indicate that the stresses to the muscle/tendonjunction are immediate and ongoing over 24 hours and different in timing to the response tothe injury within the muscle fibre. This may suggest tissue specificity as a result ofexercise-induced muscle injury. Although it has been suggested that there is both musclefibre and connective tissue damage following eccentric exercise (4), it is not known in whatproportion they occur or how they relate to each other or how injury to each tissue affectsthe symptoms of exercise-induced muscle injury.In the study outlined in Chapter Two intensity of soreness and unpleasantnessreached their highest levels at 24 hours post-exercise while range of motion (ROM) had thegreatest loss at 24 hours and eccentric torque declined for the second time at 24 hours postexercise. Tt would be preliminary to conclude that the presence of Tc-99m WBC wasrelated to the changes in the other outcome measures because Tc-99m WBC data was notcollected beyond 24 hours and the Tc-99m WBC data was not correlated with the otheroutcome measures. But the results indicate directions for future study.93In both studies the Descriptor Differential Scale (DDS), which assessed more thanone dimension of discomfort, was utilized. Using the DDS, the subjects’ perception ofintensity of soreness as well as their perception of unpleasantness of soreness - theaffective or emotional response to discomfort - was examined. The results suggest thatalthough the affective domain was not a primary experience for the subjects, in that thehighest unpleasantness response was lower than that of the intensity of soreness, the twodimensions were related. Taking into consideration the different sample sizes, there wereonly two test times (at 20 and 24 hours in Chapter Two) when the two measures were notsignificantly correlated.The results of the statistical analysis of creatine phosphokinase (CPK) indicated thatthere were no significant differences among the measures even though there was a largedifference in the means at 96 hours after the exercise stimulus. The power of the analysiswas 0.478 suggesting a 52% probability of a Type II error, or the statistical conclusion thatthere is no difference when in reality there is a difference. The pattern of the responses wassimilar to what has previously been reported in the literature and there were significantcorrelations with intensity of soreness and unpleasantness. The additional informationcontributed by this examination of patterns and relationships, together with the poweranalysis, cautions the investigator not to dismiss findings because the outcomes were notstatistically significant. In this case, the lack of significance was most likely due to thecharacteristic inter-subject variability of CPK.The cause of delayed muscle soreness following eccentric exercise is not known.The unpleasantness response of the DDS was significantly correlated with CPK at four andseven days post-exercise and ROM at 20 hours post-exercise. There were also significantcorrelations between intensity of soreness and CPK at four and seven days post-exercise.94The relationship between loss of ROM and unpleasantness suggests that pressure fromswelling stimulates sensory nerve endings. However, the relationship between CPK andeither intensity of soreness or unpleasantness does not appear as obvious, unless therelationship is reflecting the time it takes for CPK to reach the circulation through theinterstitium. The greater the amount of swelling and damage to the connective tissue (5),and thus discomfort, the longer it takes for CPK to reach the circulation.As investigators attempt to understand the mechanisms of muscle injury, the causeof muscle soreness, and the subsequent repair of muscle tissue, greater knowledge isobtained about the muscle at many levels. This knowledge is also being applied to thestudy of muscle diseases. As well, investigations concerning the morphological andultrastructural changes in the muscle in response to eccentric exercise and overload areproviding information about the way the contractile component functions to produceeccentric activity. As the mechanism by which muscle generates force is still mostly basedon theory (6) these are important contributions to the study of muscle.Future research from these studies may focus on radionuclide labelling of specificinflammatory cells such as neutrophils, macrophages and lymphocytes in order to betterdocument the time course of the inflammatory process in muscle after eccentric exercise.These techniques may then be applied to the study of the response of debilitated muscle toexercise after prolonged immobilization.95References1. Faulkner JA, Brooks SV, Opiteck JA. Injury to skeletal muscle fibres duringcontractions: conditions of occurrence and prevention. Physical Therapy 1993 ;73( 12):91 1-921.2. Armstrong RB. Initial events in exercise-induced muscular injury. Medicine andScience in Sports and Exercise 1990 ;22(4):429-435.3. Clarkson PM, Nosaka K, Braun B. Muscle function after exercise-induced muscledamage and rapid adaptation. Medicine and Science in Sports and Exercise1992 ;24(5) :512-520.4. Stauber WT, Clarkson PM, Fritz VK, Evans WJ. Extracellular matrix disruptionand pain after eccentric muscle action. Journal of Applied Physiology 1990;69(3):868-874.5. Nosaka K, Clarkson PM, Apple FS. Time course of serum protein changes afterstrenuous exercise of the forearm flexors. Journal of Laboratory Clinical Medicine1992;119(2): 183 - 188.6. Chapman AE. Exercise and Sport Science Reviews.Baltimore: Williams andWilkins, 1985:443-501. The mechanical properties of human muscle.96APPENDIX ONEReview of the literatureSkeletal muscle is one of the most adaptable tissues in the body (1). Whether theactivity level increases or decreases, the muscle responds continuously to the changes in itsinternal and external environment (2). It was commonly believed, until recently, thatskeletal muscle fibres were unable to repair themselves after injury or disease, but now theability of skeletal muscle to regenerate after injury is recognized (2) and the similarity of theprocess to the embryonic development of muscle has been observed (1).Following serious injuries or prolonged immobilization, recovery of completeendurance and strength is often a slow and arduous task. This may be related to the abilityof the debilitated skeletal muscle to respond to exercise, however, much of it may be relatedto a trial and error approach to exercise progression based on the patient’s feedback. If thetraining intensity is too small, progression will be much slower than necessary. However,if the training intensity is too severe, fatigue may persist and there may be alterations in themuscle consisting of cellular and structural damage.Although some cell death and turnover may be an important component of the processoptimizing the adaptation of skeletal muscle’s response to training, significant lethal celldamage may severely hinder recovery. The ability of skeletal muscle to regenerate afterdamage has been well established in mammalian species (3), however, it has beenestimated that repair of damaged muscle may take as long as twelve weeks (4). Healthyindividuals who had run a marathon demonstrated ultrastructural changes in theirgastrocnemius indicative of degeneration and regeneration of skeletal muscle eight and ten97weeks following the race (5). From animal studies, it would appear that exercise facilitatesalignment of normal architecture and optimal metabolism in muscle (2, 6).Delayed onset muscle soreness (DOMS) is a sensation of discomfort associated withmovement or palpation usually felt in skeletal muscle 24 to 72 hours followingunaccustomed muscular exertion (7). Four major hypotheses have been put forth as beingthe causative factor of DOMS: high tension per unit area of muscle resulting in structuraldamage, increased metabolism resulting in the accumulation of waste products, increasedtemperature that causes structural damage, and altered neural control resulting in musclespasm (7). Structural damage from high tension is the hypothesis that has received themost support to date. As early as 1902, Hough (8) described two distinct types of painassociated with exercise: pain which accompanies high intensity exercise likely related tometabolic or circulatory factors, and pain arising after the exercise (which may be causedby breaking of adhesions formed during the repair process resulting from rupture ofcontractile and connective tissue elements in the muscle).The muscle activity which causes the most soreness and the most damage is eccentricactivity (9, 10). During eccentric activity, the force developed is approximately twice thatdeveloped during isometric contractions. The lowest forces are developed duringconcentric contractions. The total number of attached cross bridges in a strongly boundstate during eccentric activity is only about 10% greater than during an isometric contraction(11). Therefore, it seems probable that the mechanism of injury from eccentric exercise isdue to the increased tension per individual cross bridge (11) causing mechanical disruptionof the ultrastructural elements within the muscle fibres such as the Z-band and contractilefilaments (12). Stauber and colleagues (13) have suggested that DOMS is due to a complexset of reactions involving disruption of the muscle fibre and connective tissue. The specificmechanism leading to DOMS, however, is still poorly understood.98Muscle soreness, or DOMS, is one of the characteristics of contraction-induced orexercise-induced muscle damage. Other characteristics include swelling, loss of musclestrength, and changes in the biochemical markers of the muscl&s integrity (14). Smith(15) has suggested that the sensation of muscle soreness, swelling and loss of function arealso associated with the acute inllammatoiy response. This theory was initially proposed inthe 1970’s but at that time was not supported by research findings. Similar to Stauber andcolleagues (13), Smith (15) believes that mechanical disruption to the muscle fibre andconnective tissue is a result of the unaccustomed eccentric exercise, but she goes on topoint out that within a few hours of the injury the acute inflammatory response begins.First white blood cells migrate to the area, including neutrophils in the first few hours andmonocytes 6-12 hours after the neutrophils. When the monocytes enter the tissuecompartment from the blood, they mature into macrophages. The macrophages are at theirgreatest numbers at 48 hours. Their function is to remove the dead tissue but they are alsoimportant in the healing process. Smith (15) suggests that the presence of macrophagesmay be responsible for the synthesis of prostaglandins which may be related to thesensation of soreness at 48 hours.It is not known whether decreased ROM reflects shortening of the contractilemechanism (16), increased edema within the connective tissue resulting in increasedpressure (17), or injury to the connective tissue (14) or a combination of all three.Evidence of damage to the connective tissue is limited (10). Abraham (18) reportedelevated hydroxyproline, a component of collagen, after exercise which included eccentricactivity. However, few other studies have confirmed his results. Fritz and Stauber (19)showed noticeable histological variation by 24 hours in the proteoglycan component of theextracellular matrix (ECM) of rat muscle following eccentric activity. They suggested thatstructural disruption of the proteoglycan component may result in attraction of water within99the ECM as part of an osmotic force that leads to fluid accumulation. Clarkson andcolleagues (14) have reported that changes in the circumference of the upper arm aftereccentric exercise of the elbow flexors peaked at five days post-exercise while changes inthe relaxed elbow angle (the angle at the elbow when the arm hangs freely by the side) weregreatest at three days. Therefore, they believe that swelling and changes in ROM are notrelated. As Stauber (10) has commented, further research is necessary to support thehypothesis of connective tissue damage after eccentric exercise.After fatiguing eccentric exercise, there is an immediate decrease in maximal forceproduction which has been observed as early as one hour after the exercise (12, 20, 21).Newham and colleagues (22) reported that strength returned to the pre-exercise levelswithin 24 hours but others have found that it has taken as long as a week (21) or more(11). Assessing the relationship between development of soreness and the loss of musclestrength suggests that there is little or no relationship between the two (16). Thus, it seemsunlikely that muscle soreness contributes significantly to the loss of muscle force.It is not known if the initial decline in strength is due to fatigue or muscle injury.Clarkson and colleagues (14) have proposed that the immediate loss of strength may be dueto overstretched sarcomeres in which the overlap between actin and myosin filamentswould be reduced, thereby affecting force production. The fact that Newham andcolleagues (23) have found greater strength losses after eccentric exercise at a long musclelength compared to a short muscle length lends support to this hypothesis.Faulkner and colleagues (11) have reported a biphasic response in the muscle force inanimals after eccentric exercise. They believe that the initial decline in force may be afunction of mechanical injury and fatigue, especially where the subjects have just completedan exhaustive eccentric protocol. There is supporting evidence in the literature of100myofibrillar disruption at the level of the Z-line after eccentric exercise. Armstrong andcolleagues (24) reported disruptions in the striation pattern of slow twitch fibres of theextensor muscles in rats immediately following downhill running. Friden and colleagues(25) identified disorganization of the Z-line in human soleus muscle three days afterdownhill running. Newham and colleagues (26) showed that extensive sarcomericdisruptions had occurred in human quadriceps muscles immediately after eccentriccontractions. Areas of damage have been observed immediately after exercise withcontinuing disruption to the Z-line over the next few days (27).Faulkner and colleagues (11) go on to suggest that a secondary injury occurs to themuscle and that it is a biochemical one as a result of the phagocytic activity at the site of theoriginal damage. Neutrophils and monocytes release oxygen radicals, potentially causingfurther damage to the muscle. When monocytes enter the tissue they mature intomacrophages (15). Animal studies have provided evidence of the presence of macrophagesin the injured muscle after eccentric exercise (24) but human morphological studies havenot provided firm evidence of the acute phase response in the exercised muscle within 48hours of eccentric exercise (25, 28). Evans and Cannon (29) believe that the absence ofmorphological evidence in human studies is mostly due to sampling procedures which missthe region of injury or timing that is later than the acute phase response.Some investigators have assessed products released in the urine, blood or plasmafrom the breakdown of connective tissue or muscle, or the increased permeability of themuscle fibre. Of these techniques, most of the studies to date have investigated therelationship between DOMS and muscle products such as creatine phosphokinase (CPK),lactate dehydrogenase or myoglobin (7, 18, 28, 30-35). One of the most commonlystudied serum proteins is the response of CPK after exercise (36). CPK, a marker forsome muscle diseases (14), has been shown to increase after eccentric exercise but it does101not have the same time course as the perception of soreness (13). It usually begins toincrease by 24 hours after the eccentric exercise and reaches its highest values three to sixdays post-exercise (36). Other muscle proteins, such as lactate dehydrogenase andmyoglobin, show a similar delay in their increase in the blood after eccentric exercise (14).CPK is a key enzyme in the ADP - ATP transformation (37). It is released from themuscle tissue into the interstitium and then it travels to the circulation via the lymphaticsystem (16). It is not known what mechanism mediates the release of CPK from thetissue. Evans and Cannon (29) have stated that the post-exercise CPK response is amanifestation of muscle damage but not a direct indicator of it.An early investigation by Abraham (18) examined a marker of connective tissuedamage, the appearance of urine hydroxyproline (OHP). Further support of the importanceof connective tissue damage contribution to muscle soreness and poor performance comesfrom a more recent study where evidence of the disruption of extracellular matrix occurredat the same time as an increase in muscle soreness (13). Essentially all OHP in vertebratetissue is found in collagen, except for a small amount in elastin (38). This uniquedistribution of OHP makes it a useful label for studying the metabolism of collagen (38).Other investigators have also reported on OHP after exercise. Seaman and lanuzzo(34) reported a lack of significant change in the hydroxyproline/creatimne (OHP/Cr) ratioand serum Ol-IP following 30 minutes of double leg extensions. Horswill and colleagues(39) also reported a lack of significant change in the OHP/Cr ratio and OHP excretionfollowing three circuits of nine exercises on a Universal Gym. However, Murguia and102colleagues (40) reported a significant baseline increase in the plasma OHP values of NAVYSEAL candidates who later developed connective tissue injuries during their trainingprogram, compared to candidates without injury. In other words, those at risk forconnective tissue injuries had elevated OHP levels at the beginning of training.The most direct evidence of muscle damage is provided by ultrastructural andhistological analyses of muscle damage. Friden and colleagues (12) have shown that thereis broadening, streaming and sometimes total disruption of the Z-bands following eccentricexercise. They noted that these changes continued to increase up to three days after theinsult but by six days the evidence of muscle damage had reduced. They have also notedchanges in the sarcolemma (12). Some authors have suggested there may be an associationbetween the damaged sarcolemma and increases in levels of calcium ions (22, 41). Thelevels of calcium ions are important in both inducing muscle damage and in the repairprocess but Ebbeling and Clarlcson (16) have cautioned that much more investigation isneeded to understand the effect of altered levels of calcium ions on the muscle. Stauber andcolleagues (13) observed separation of the extracellular matrix, mast cell degranulation, andincreased plasma constituents in the extracellular space in biopsies taken at 48 hours. Theysuggested that the muscle soreness also seen at 48 hours is a result of the inflammatoryresponse which they believe is in response to the extracellular disruption. However, intheir study biopsies were taken at only one time period (48 hours).Although there has been considerable success in producing an exercise model whichreproduces the contraction-induced injury in skeletal muscle, the mechanism of the injury isstill not known. Most researchers believe that the initiating event is a mechanical one butthe possibility of a metabolic component contributing to the injury has also been suggested(11). Faulkner and colleagues (11) have suggested that during lengthening of the muscle,some sarcomeres maintain their length while others are stretched beyond overlap and103injured. Lieber and Fnden (42) have proposed that the cytoskeleton of muscle is the firststructure to yield after eccentric activity, followed by myofibrillar disruption of the A-bandand the Z-band. They have also suggested that, because of the damage to the Type II fibresin human muscle, fibre oxidative capacity might be an important factor in determining theextent of fibre damage following eccentric exercise (43). Perhaps the Type II fibres fatigueearly in the exercise; they are unable to regenerate ATP and subsequently enter a “stiffness”state. These stiff fibres are then stretched and damaged during the eccentric activity.Clarkson and colleagues (14) have also proposed that there may be stiffness in the crossbridge but they believe that it occurs for a few days after the exercise as a result ofincreased intracellular calcium (Ca++). Armstrong (41) has also proposed that as a resultof injury to the sarcolemma there is a loss of Ca++ homeostasis in the muscle. This stageof events occurs before the inflammatory cellular response and begins the degradativeprocess through activation of phospholipase A2 in the cell membrane and the subsequentleukotriene and prostaglandin cascades.The challenge is in determining the underlying mechanism of injury in an intactspecies. Exercise is described as promoting strengthening of the connective tissue harnessof muscle and through this mechanism is thought to be a preventive therapy for injury tothese structures. However, if the exercise stimulus exceeds the ability of the skeletalmuscle tissue to respond then DOMS, and muscle fibre and connective tissue damage willoccur. An optimal training level is achieved when the intensity of the exercise programstimulates new muscle tissue growth but minimizes excessive muscle damage. Thus, theobjectives of these studies were to characterize the time course and relationships of outcomemeasures of muscle injury after eccentric exercise and to examine the presence ofinflammatory cells in the exercised muscle.104References1. Lieber RL Skeletal Muscle Structure and Function.Baltimore: Williams andWilkins, 19922. Carison BM. Regeneration of entire skeletal muscles. Federation Proceedings1986;45: 1456-1460.3. Carlson BM, Faulkner JA. The regeneration of skeletal muscle fibres followinginjury: a review. Medicine and Science in Sports and Exercise 1983;15: 187- 198.4. Evans WJ. Exercise-induced skeletal muscle damage. The Physician andSportsmedicine 1987; 15( 1)(Jan) :88-100.5. Warholl MJ, Siegal AJ, Evans WJ, Silverman LM. Skeletal muscle injury andrepair in marathon runners after competition. American Journal of Pathology1985;1 18:33 1-339.6. White TP. Adaptations of skeletal muscle grafts to chronic changes of physicalactivity. Federation Proceedings 1986;45: 1470-1473.7. Armstrong RB. Mechanisms of exercise-induced delayed onset muscular soreness:a brief review. Medicine and Science in Sports and Exercise 1984;16(6):529-538.8. Rough T. Ergographic studies in neuro-muscular fatigue. American Journal ofPhysiology 1902;7:76-92.1059. McCully KK, Faulkner JA. Injury to skeletal muscle fibers of mice followinglengthening contractions. Journal of Applied Physiology 1985;59( 1): 119-126.10. Stauber WT. Exercise and Sport Science Reviews.Baltimore: Williams andWilkins, 1989(vol 17): 157-185. Eccentric action of muscles: physiology, injury, andadaptation.11. Faulkner JA, Brooks SV, Opiteck JA. Injury to skeletal muscle fibres duringcontractions: conditions of occurrence and prevention. Physical Therapy 1993 ;73( 12):91 1-921.12. Fridén J, Sjostrom M, Ekblom B. Myofibrillar damage following intense eccentricexercise in man. International Journal of Sports Medicine 1983;4: 170- 176.13. Stauber WT, Clarkson PM, Fritz VK, Evans Wi. Extracellular matrix disruptionand pain after eccentric muscle action. Journal of Applied Physiology 1990;69(3):868-874.14. Clarkson PM, Nosaka K, Braun B. Muscle function after exercise-induced muscledamage and rapid adaptation. Medicine and Science in Sports and Exercise1992 ;24(5) :512-520.15. Smith L. Acute inflammation: The underlying mechanism in delayed onset musclesoreness? Medicine and Science in Sports and Exercise 1991;23:542-551.16. Ebbeling CB, Clarkson PM. Exercise-induced muscle damage and adaptation.Sports Medicine 1989;7:207-234.10617. Jones DA, Newham DJ, Clarkson PM. Skeletal muscle stiffness and painfollowing eccentric exercise of the elbow flexors. Pain 1987;30:233-242.18. Abraham WM. Factors in delayed muscle soreness. Medicine and Science inSports 1977;9:11-20.19. Fritz VK, Stauber WT. Characterization of muscles injured by forced lengthening.II. Proteoglycans. Medicine and Science in Sports and Exercise 1988;20(4):354-361.20. Clarkson PM, Tremblay I. Exercise-induced muscle damage, repair, and adaptationin humans. Journal of Applied Physiology 1988;65: 1-6.21. Newham DJ, Jones DA, Clarkson PM. Repeated high force eccentric exercise:Effects on muscle pain and damage. Journal of Applied Physiology 1987;63: 1381-1386.22. Newham DJ, Mills KR, Quigley BM, Edwards RHT. Pain and fatigue afterconcentric and eccentric contractions. Clinical Science 1983 ;64:55-62.23. Newham DJ, Jones DA, Ghosh G, Aurora P. Muscle fatigue and pain aftereccentric contractions at long and short length. Clinical Science 1988;74:553-557.24. Armstrong RB, Ogilvie RW, Schwane JA. Eccentric exercise-induced injury to ratskeletal muscle. Journal of Applied Physiology 1983; 54:80-93.25. Fndén J, SjOstrOm M, Ekblom B. A morphological study of delayed musclesoreness. Experientia 1981 37:506-507.10726. Newharn DJ, McPhail G, Mills KR, Edwards RHT. Ultrastructural changes afterconcentric and eccentric contractions of human muscle. Journal of The NeurologicalSciences 1983;61: 109-122.27. Newham DJ. The consequences of eccentric contractions and their relationship todelayed onset muscle pain. European Journal of Applied Physiology 1988;57:353-359.28. Jones DA, Newham DJ, Round JM, Tolfree SET. Experimental human muscledamage: morphological changes in relation to other indices of damage. Journal ofPhysiology 1986;375:435-448.29. Evans WJ, Cannon JG. The metabolic effects of exercise-induced muscledamage.Baltimore: Williams and Wilkins, 1991:99-125. Exercise and Sports ScienceReviews30. Byrnes WC, Clarkson PM, White JS, Hsieh SS, Frykman PN, Maughan RJ.Delayed onset muscle soreness following repeated bouts of downhill running. Journal ofApplied Physiology 1985;59(3):710-715.31. Clarkson PM, Byrnes WC, McCormick KM. Turcotte LP, White JS. Musclesoreness and serum creatine kinase activity following isometric, eccentric and concentricexercise. International Journal of Sports Medicine 1986;7(3): 152-155.32. Newham DJ, Jones DA, Tolfree SE, Edwards RH. Skeletal muscle damage: astudy of isotope uptake, enzyme efflux and pain after stepping. European Journal ofApplied Physiology 1986;55(1): 106-112.10833. Schwane JA, Williams JS, Sloan JH. Effects of training on delayed musclesoreness and serum creatine kinase activity after running. Medicine and Science in Sportsand Exercise 1987; 19(6):584-590.34. Seaman R, lanuzzo CD. Benefits of short-term muscular training in reducing theeffects of muscular over-exertion. European Journal of Applied Physiology1988 ;58(3):257-261.35. Thomas BD, Motley CP. Myoglobinemia and endurance exercise: A study oftwenty-five participants in a triathion competition. American Journal of Sports Medicine1989;12(2): 113-119.36. Nosaka K, Clarkson PM, Apple FS. Time course of serum protein changes afterstrenuous exercise of the forearm flexors. Journal of Laboratory Clinical Medicine1992;119(2): 183 - 188.37. Hortobágyi T, Denahan T. Variability in creatine kinase: methodological, exercise,and clinically related factors. International Journal of Sports Medicine 1989; 10(2):69- 80.38. Prokop DJ, Kivirikko KI. Relationship of hydroxyproline excretion in urine tocollagen metabolism. Annals of Internal Medicine 1967;66(6): 1243- 1266.39. Horswill CA, Layman DK, Boileau RA, Williams BT, Massey BH. Excretion of 3-methylhistidine and hydroxyproline following acute weight-training exercise. InternationalJournal of Sports Medicine 1988;9(4):245-248.10940. Murguia MJ, Vailas A, Mandelbaum B, eta!. Elevated plasma hydroxyproline Apossible risk factor associated with connective tissue injuries during overuse. AmericanJournal of Sports Medicine 1988;16(6):660-664.41. Armstrong RB. Initial events in exercise-induced muscular injury. Medicine andScience in Sports and Exercise 1990;22(4):429-435.42. Lieber RL, Friden J. Muscle damage is not a function of muscle force but activemuscle strain. Journal of Applied Physiology 1993 ;74(2):520-526.43. Fridén J, Lieber RL. Structural and mechanical basis of exercise-induced muscleinjury. Medicine and Science in Sports and Exercise 1992;24(5):521-530.110APPENDIX TWODescriptor Differential Scale (DDS)I Instructions to the SubjectsFigures 11 and 12 are examples of the intensity of soreness scale and theunpleasantness scale respectively. Subjects scored their intensity of soreness andunpleasantness immediately following the test of eccentric torque, which was meant to bethe reference activity for scoring the DDS. Subjects scored the DDS on a computer wherethe descriptors were generated onto the screen one at a time and in a randomized order.The subjects were instructed to think about their soreness relative to each anchoringdescriptor. If the descriptor perfectly described their soreness, then they were instructed tomark underneath it; if their soreness was proportionally less than that described by thedescriptor then they were to mark to the left of the descriptor; and if their soreness wasproportionally more than that described by the descriptor they were to mark to the right ofthe descriptor (1).II CalculationsThe 12 descriptors were placed in order of increasing descriptor magnitudeaccording to Gracely and Kwilosz (1). Each descriptor was scored from -10 to +10. Thedata for each scale was plotted with the descriptors on the X axis and the scores on the Yaxis. In an attempt to represent the increasing magnitude of the descriptors, the intercept ofthe line of best fit through the 12 data points was used as the score of each scale. Figure 13is an example of one subject’s soreness response at 24 hours post-exercise. Thedescriptors on the X axis are ranked in order of increasing magnitude. Figure 14 is an111Figure 11. Amount of Sensation. The 12 descriptors of the intensity of soreness scale arepresented in randomized order. See text for explanation.112Figure 11PART A - Amount of SensationRate your sensation in relation to each woni with a check mark.Slightly Intense- +Mild- +Strong- +Faint- +Veiy Weak- +Very Intense- +Moderate- +Veiy Mild- +Weak- +Extremely Intense- +Barely Strong- +Intense- +113Figure 12. Amount of Unpleasantness. The 12 descriptors of the unpleasantness scale arepresented in randomized order. See text for explanation.114PART B- Amount of Unpleasantness Figure 12Rate your unpleasantness in relation to each word with a check mark.Very Unpleasant- +Very Distressing- +Slightly Unpleasant- +Unpleasant- +Slightly Intolerable- +Annoying- ÷Distressing- +Slightly Annoying- +Slightly Distressing- +Very Intolerable- +Intolerable- +Very Annoying+115Figure 13. Subject 1: Intensity of Soreness at 24 Hours Post-Exercise. On the y axis isthe score for each descriptor for this subject. The descriptors are listed on the xaxis in order of increasing magnitude.QL.flLfl00_______________-SIIICDFAINTVERYWEAK.WEAK•VERYMILD.MILDMODERATEBARELYSTRONGSLIGHTLYINTENSESTRONG.INTENSE•VERYINTENSEEXTREMELYINTENSE•U’I-a117Figure 14. Subject 1: Unpleasantness Response at 96 Hours Post-Exercise. Thedescriptions of the y axis and x axis are the same as Figure 13. Note that differentwords have been chosen as descriptors for this scale.-oViVi’,—s CDSLIGHTLYUNPLEASANT.SLIGHTLYANNOYINGUNPLEASANTANNOYINGSLIGHTLYDISTRESSING‘‘I•VERYUNPLEASANT.DISTRESSINGVERYANNOYING.SLIGHTLYINTOLERABLEVERYDISTRESSING•INTOLERABLEVERYINTOLERABLEUiI-.119example of the same subject’s unpleasantness response at 96 hours post-exercise, with thedescriptors in order of increasing magnitude on the X axis.III Reliability and ValidityPurposeThe purpose of this pilot study was to assess the test-retest reliability andconcurrent validity of the intensity of soreness and the unpleasantness scales of the DDS insubjects with DOMS. Gracely and Kwilosz (1) have previously reported the DDS to bereliable in a group of 91 dental patients.ProtocolSeven subjects (four females and three males), ages 23 to 34 years, participated in apilot study of the test-retest reliability of the DDS. Two of those subjects did not participateat 96 hours and 168 hours post-exercise. The subjects scored the DDS at the usual testingtimes - pretest, and 24 hours, 48 hours, 96 hours and 168 hours following the eccentricexercise (test 1). They returned one hour later, at each test session, and following fourrepetitions of the eccentric torque protocol, they scored the DDS again (test 2).Eighteen subjects (14 females and four males) ages 20 to 52 years, scored theirsoreness according to the Visual Analogue Scale (VAS) in addition to the DDS. The VAShas been utilized in other studies of delayed onset muscle soreness (2). The DDS scores,over the five testing times, were correlated with the scores of the VAS to determineconcurrent validity.120Data AnalysisPearson product-moment correlation analyses were used to determine test-retestreliability between test 1 and test 2, and concurrent validity between the DDS (intensity ofsoreness and unpleasantness) and the VAS.ResultsTable 12 summarizes the test-retest reliability and the concurrent validity of the DDS.The test-retest reliability of the intensity of soreness ranged from 0.72 to 0.99 over the fivetest times. For the unpleasantness scale the correlations ranged from 0.64 to 0.98, exceptfor the the pre-test day (r=0.58). On the pre-test day five out of seven subjects had thesame scores (-10) on both test occasions. One subject had scores that were -9 at bothtimes, and the other subject’s second score was within one point of the first score (-10).However, as these responses caused a clumping of the scores, and the Pearson r is ameasure of linearity, the correlation was poor even though the scores were in fact veryclose over the two test times.Richman and colleagues (3) have suggested a scheme to rank the correlation values -0.80 to 1.0 is “very reliable”; 0.60 to 0.79 is “moderately reliable” and 0.59 and less has“questionable reliability”. All of the intensity of soreness correlations for test-retestreliability would be considered very reliable except at 24 hours post-exercise, which wouldbe considered moderately reliable. Two of the unpleasantness correlations would betermed very reliable and two would be considered moderately reliable. The pre-testcorrelation appears of questionable reliability but in fact the scores were very repeatable.121TABLE 12PEARSON PRODUCT-MOMENT CORRELATION COEFFICIENTS FOR THEPre-testn=70.92DESCRIPTOR DIFFERENTIAL SCALETest-Retest Reliability for Intensity of Soreness24Hrn=70.72Testl48Hrn=70.8596Hrn=50.80Test-Retest Reliability for UnpleasantnessTest 1168Hrn=50.99Pre-test 24Hr 48Hr 96Hr 168Hrn=7 n=7 n=7 n=5 n=5Test2Pre-test 0.5824Hr 0.7448Hr 0.9796Hr 0.98168Hr 0.64Concurrent ValidityVASIntensity of Soreness 0.84Unpleasantness 0.63Test2Pre-test24Hr4SHr96Hr168Hr122The concurrent validity of the soreness scale of the DDS with the VAS for 18subjects over two exercise durations and five test times was 0.84, while the validity of theunpleasantness scale with the VAS was 0.63. The higher correlation between the intensityof soreness scale and the VAS suggested that subjects scored the VAS relative to intensityof soreness.These results suggest that the DDS is an objective measure of muscle soreness.They also substantiate the findings of Gracely and Kwilosz (1) that the DDS is a reliableinstrument.123References1. Gracely RH, Kwilosz DM. The descriptor differential scale: applyingpsychophysical principles to clinical pain assessment. Pain 198835:279-288.2. Bobbert MF, Hollander AP, Huijing PA. Factors in delayed onset muscularsoreness of man. Medicine and Science in Sports and Exercise 1986;18(1):75-81.3. Richman J, Makrides L, B P. Research methodology and applied statistics, Part 3:Measurement procedures in research. Physiotherapy Canada 1980;32: 253-257.124APPENDIX THREEPower Spectrum AnalysisI CalculationsThe force and raw EMG signals were simultaneously collected by the computer forspectral analysis. The sampling rate was 500 Hz. Figure 15 illustrates two sections of theraw data from six to nine seconds and from 54 to 57 seconds respectively for one subject.As the subject fatigued and force declined, the EMG amplitude declined and there weremore areas of the EMO that were relatively inactive (Figure 15 B) compared to the earliersignal (Figure 15 A).Spectral analysis entailed partitioning the EMG data into overlapping four-secondsegments with each successive segment starting two seconds later than the previoussegment. Spectral estimates of each four-second segment were then calculated through aFast Fourier Transformation. Figure 16 is an example of the spectral frequency for thevastus lateralis (VL), rectus femoris (RF) and vastus medialis (VM) muscles for one four-second segment. Lindstrom and Magnusson (1) have reported that as muscle fatigues thespectral frequency shifts to a lower frequency indicating a decrease in action potentialconduction velocity. Other investigators suggest that although there is a relationshipbetween spectrum analysis and conduction velocity, factors such as the rate of motor unitfiring also contribute to the change in EMG signal frequency and thus, muscle fatigue (2).The median frequency, which was calculated from the area under the curve, served asthe measure by which muscle fatigue was calculated. The median frequency of each foursecond segment was plotted against time and a linear regression line was fitted to the 28125Figure 15. Force and Raw Electromyography (EMG) Signals. A. Recordings between 6and 9 seconds. B. Recordings between 54 and 57 seconds. VL is the vastuslateralis muscle, RF is the rectus femons muscle, and VM is the vastus medialismuscle.126A Figure 1514000 I12000 VL100008000 RF60004000 ‘VM20000-- Force6 6:5 7.5 8 8.5 9Time (seconds)B14.c)cX) — I12000 k4k*M ft VL10000-8000 RF.6000-4000 VM20000-Force54.5 56 56.5 57Time (seconds)127Figure 16. Typical Surface-EMG Power Spectrum. VL=vastus lateralis, RF=rectusfemons, VM=vastus medialis. See text for explanation.Figure 16Frequency (Hz.)128Typical Surface-EMO Power Spectrum4 4 I4low10Ut 104.2 101061O::::7:J::::::::::::::.: :::::::::::::::::::::::::::::: :‘‘—“.“::: ::::::Ib.JI5.Q.%, .:::::::2;::::::::]::::::::::.:::::.:::::::::::::ft \4\_%_4VLRFWI0 50 100 150 200 250129Figure 17. Regression Lines for the Frequency Spectra. VL=vastus lateralis, RF=rectusfemoris, VM=vastus medialis. “Half-Power” is the Median Frequency. See textfor explanation.Figure 17II I600C)I_ nIs4 --IU004nfl20IC130100yu.RFVL, WI10 20 30 40 50 60Time (seconds)131data points (Figure 17). The slope of the line was considered a quantitative measure ofmuscle fatigue. The steeper the negative slope of the line, the greater the muscle fatigue.Other investigators have sampled data at the beginning, middle and end of a contraction (3)or every 10 seconds throughout the contraction (4). By analyzing 60 seconds of data, inoverlapping segments, 28 times, a more precise reflection of the changes in the frequencywas achieved.II Reliabffity and ValidityPurposeThe purpose of this pilot study was to determine if the power spectrum analysis isan objective measure of muscle fatigue.ProtocolSix subjects (three females and three males), from 20 to 43 years, participated in astudy of test-retest reliability and concurrent validity of the power spectrum analysis. Thesubjects repeated the protocol on two separate days not more than one week apart. Theslope of the median frequency of each muscle was compared to the slope of isometric forceto determine concurrent validity. Bigland-Ritchie (5) has stated that loss of forcecharacterizes muscle fatigue.MethodsThe subjects were seated on the KinCom isokinetic dynamometer, with their hips at80 degrees, their back supported and the pelvis stabilized on the bench. The centre of132rotation of the KinCom was positioned opposite the centre of the knee joint line. Theresistance pad was positioned at a point on the lower leg that was 75% of the length of thefibula. The knee was positioned at 90 degrees of knee flexion.The skin was cleaned with an alcohol swab and surface EMG electrodes (MediTrace silver/silver chloride, circular, 1 cm radius) were placed over the motor points ofVL, RF, and VM muscles according to Delagi and colleagues (6). The interelectrodespacing was 2.5 cm. A ground electrode was placed over the wrist. Electrodes wereplaced on the skin with electromedical gel between the skin and the electrodes. Subjectsmaintained a maximum isometric contraction of the knee extensors for 60 seconds. Aten-second submaximal isometric contraction preceded the one minute fatiguingcontraction as a warm-up.Data AnalysisPearson product-moment correlation analyses were used to assess test-retestreliability of the slope of the median frequency for each muscle between day 1 and day 2,and concurrent validity between the slope of the median frequency of each muscle and theslope of the isometric force over the two test times. Isometric force, recorded every twoseconds over the 60 seconds of the test, was plotted against time and a linear regressionline was fitted to the data points. The slope of the line was the quantitative measure of thechange in force.ResultsTable 13 summarizes the results of test-retest reliability and the concurrent validityof the power spectrum analysis. All of the correlations between day 1 and day 2 for the133TABLE 13POWER SPECTRUM ANALYSIS:PEARSON PRODUCT-MOMENT CORRELATION COEFHCIENTSTest-Retest ReliabilityVL1* RF1 VM1VL2 0.99RF2 0.90VM2 0.98* VL1, VL2 are the Median Frequencies of Vastus Lateralis at Test 1 and Test 2respectively. RF1, RF2 are the Median Frequencies of Rectus Femons at the same testtimes, and VM1, VM2 are the Median Frequencies of Vastus Medialis at the same testtimes.Concurrent ValidityForceMedian Frequency of Vastus Lateralis 0.88Median Frequency of Rectus Femons 0.91Median Frequency of Vastus Medialis 0.89134slopes of the median frequency of each of the muscles were greater than 0.90. Thecorrelations between the slope of the median frequency of each muscle and the slope ofthe isometric force were all above 0.87.These results suggest that the power spectrum analysis is a reliable and valid toolfor quantifying muscle fatigue.III Fatigue of the Rectus Femoris MuscleFigure 18 is a graph of the mean slope of the median frequency of RF, plus andminus one standard deviation, for the ten subjects who participated in the study reported inChapter Two. Contrary to the responses of VL and VM, the slope of the median frequencyof RF increased following the eccentric exercise protocol. The results suggested that themuscle was not fatigued but this seemed unlikely after 300 eccentric contractions over 30minutes. The response of RF was also different to the responses of the other twosuperficial quadriceps muscles even though all of the muscle are knee extensors. Furthertesting was conducted in an attempt to understand the response of RF.As RF is a muscle that crosses both the hip and the knee joints, the position of thehip joint was changed to a neutral position. By lying subjects supine on the plinth, with thehip at 0 degrees, RF was isolated to working as a knee extensor at a longer muscle length.Five female subjects (24 - 43 years), who were part of another study, participatedin the same exercise stimulus and power spectrum analysis protocol as has been outlined inChapter Two. Data was collected before the eccentric exercise (pre-test), immediately afterthe exercise protocol (0 hour) and at two hours post-exercise for the subjects. Thesesubjects were postioned in sitting to confirm or reject the results for RF from the study135Figure 18. Power Spectrum Analysis: Rectus Femoris Median Frequency. Values aremeans+SD.SLOPEOFTHEMEDIANFREQUENCY)p-PPcU,U,0’0’Cr1[I.CDIIF1II-C.)137outlined in Chapter Two. One subject did not participate in the data collection at two hourspost-exercise. Table 14 summarizes the descriptive statistics and Figure 19 A illustrates theslopes of the median frequencies for the three quadriceps muscles - VL, RF and VM.Similar to the early post-exercise responses of the subjects who participated in thestudy reported in Chapter Two, the slopes of the median frequencies of VL and VMdeclined over two hours post-exercise and the slope of the median frequency of RFincreased.Next, nine female subjects, 21 - 47 years, participated in the same protocol exceptthat these subjects were postioned supine with the hip at 0 degrees. The knee was stillpositioned at 90 degrees of knee flexion. The instructions to these subjects were that theywere not to lift their heads or their shoulders during the testing. Data were collected beforethe exercise stimulus and at 0 hour. Table 14 summarizes the descriptive statistics andFigure 19 B represents the slopes of the median frequencies for the three muscles (VL, RF,VM) from the pre-test toO hour. With a change in the position of the hip joint the slope ofthe median frequency of RF had decreased at 0 hour compared to the pre-test.The results of these pilot studies suggest that the position of the hip joint influencedthe power spectrum analysis of RF, but for VL and VM the pattern of the response wassimilar to that reported in Chapter Two. The most obvious difference, with the change inthe hip position from 80 degrees of flexion to neutral, was that RF was working at a longermuscle length with the hip in neutral. The change in the hip joint position did not affect VLand VM as they only cross the knee joint.According to the length-tension relationship of muscle, a shortened muscle withoverlapping actin filaments within the sarcomeres, generates much less tension than at the138TABLE 14POWER SPECTRUM ANALYSIS:DESCRIPTIVE STATISTICS OF THE MEDIAN FREQUENCIES OF THEQUADRICEPS MUSCLESWITH THE HIP AT 80 DEGREES FLEXIONTest TimesPre-test 0 Hour 2 Hoursn=5 n=5 n=4VastusLateralis -0.115 (0.05) -0.199 (0.13) -0.161 (0.08)Rectus Femons -0.234 (0.13) -0.180 (0.13) -0.193 (0.06)VastusMedjalis -0.115 (0.11) -0.163 (0.08) -0.192 (0.11)Data are means (SD).WITH THE HIP AT 0 DEGREES FLEXIONTest TimesPre-test 0 Hourn=9 n=9Vastus Lateralis -0.127 (0.08)-0.258 (0.09)Rectus Femons -0.194 (0.08)-0.253 (0.07)Vastus Medialis -0.087 (0.07)-0.254 (0.11)Data are means (SD).139Figure 19. Slopes of the Median Frequencies of the Superficial Quadriceps Muscles.A. With the hip in 80 degrees of flexion. B. With the hip neutral. Values aremeans + SD. VL=vastus lateralis, RF=rectus femons, VM=vastus medialis.SLOPEOFTHEMEDIANFREQUENCYSLOPEOFTHEMEDIANFREQUENCY-I m XC-0 -‘ C’,r++-1 m 0 -‘ Cd)Iliii!)IIr.4ppPDPPcajI\j-CC_13(rI(11CDP(11C1-v CD5 CDC141resting length of the muscle (7). The exact resting length of rectus femoris in vivo is notknown, but with the hip at 80 degrees of flexion and the knee at 90 degrees of flexion themuscle may have been positioned in a relatively shortened position. If there was littletension development there would be little evidence of fatigue over 60 seconds. Lieber (7)has stated that muscle and joint relationships have not been thoroughly studied in humans.The biomechanics and force-generating properties of muscles in regard to fibre length, fibreand muscle area, tendon length and moment arms must be considered. In addition, thearchitecture of the muscle contributes to its tension-producing capacity (7). Thearrangement of muscle fibres either enhances the strength producing capabilities or theexcursion of the muscle.Further investigation of the response of RF to fatiguing exercise is required. Aresearch design in which subjects repeated the experiment twice with the order of theposition of the hip randomly assigned, with hip position as the independent variable, andincluding both male and female subjects, would determine if hip position does influence theresponse of RF during power spectrum analysis. The other possibility is that subjects mayhave rotated the hip internally or externally in sitting, selectively recruiting VL and VMrespectively, during the isometric knee extension. Strapping of the thigh to prevent internaland external rotation would eliminate this possible confounding variable.IV Fatigue of the Vastus Lateralis and Vastus Medialis MusclesThe results of the post hoc testing of the slopes of the median frequencies of VLand VM in Chapter Two showed a significant difference only between four hours and 20hours post-exercise for VL, when the muscle appeared to be recovering from fatigue.142There were no significant differences between the pre-test and two hours post-exercise foreither muscle.In this pilot study a paired t-test of the slopes of the median frequencies of the threemuscles was used to analyze the differences between the pre-test and 0 hour for the ninesubjects whose results are illustrated in Figure 19 B. These subjects were postioned withthe hip in neutral. Table 15 summarizes the results of the paired t-test. For all of themuscles the slopes of the median frequencies significantly declined from the pre-test to 0hour (p.cO.O5) These results should be interpreted cautiously, however, because the t-testonly analyzes the differences between the means (8).Although others have suggested that the muscle’s recovery from fatigue may takeone or more days (9, 10), Faulkner and colleagues (11) have proposed that most of therecovery takes place in the first three hours post-exercise. The results of these pilot studiessuggest that their observations may be correct and that muscles quickly recover even from astrenuous exercise regime.143TABLE 15POWER SPECTRUM ANALYSISPAIRED T-TESTSSource mean difference t pVastus Lateralis Median Frequency (df, 8)pre-test-Ohr 0.135 3.81 0.005*Rectus Femoris Median Frequency (df, 8)pre-test-Ohr 0.060 2.62 0.031*Vastus Medialis Median Frequency (df, 8)pre-test-Ohr 0.167 5.90 <.001** signficantly different from the pre-test.144References1. LindstrOm LH, Magnusson RI. Interpretation of myoelectric power spectra: amodel and its applications. Proceedings of the IEEE 1977;65(5):653-662.2. Zwarts MJ, Van Weerden TW, Haenen HTM. Relationship between averagemuscle fibre conduction velocity and emg power spectra during isometric contraction,recovery and applied ischemia. European Journal of Applied Physiology 1987;56:212-216.3. Frascarelli M, Rocchi L, Feola I. EMG computerized analysis of localized fatigue induchenne muscular dystrophy. Muscle and Nerve 1988;11:757-761.4. Tarkka IM. Power spectrum of electromyography in arm and leg muscles duringisometric contractions and fatigue. Journal of Sports Medicine 1984;24: 189-194.5. Bigland-Ritchie B. EMG/force relations and fatigue of human voluntarycontractions. 1981:75-117. Exercise and Sport Science Reviews6. Delagi EF, Perotto A, lazetti J, Morrison D. Anatomic Guide for theElectromyographer. Springfield, IL: C. Thomas, 19757. Lieber RL. Skeletal Muscle Structure and Function.Baltimore: Williams andWilkins, 19928. Glenberg AM. Learning From Data: An Introduction to Statistical Reasoning.NewYork: Harcourt Brace Jovanovich, 19881459. Newham DJ, Mills KR, Quigley BM, Edwards RI-IT. Pain and fatigue afterconcentric and eccentric contractions. Clinical Science 1983 ;64:55-62.10. Jones DA, Newham DJ, Torgan C. Mechanical influences on long-lasting humanmuscle fatigue and delayed-onset pain. Journal of Physiology 1989;412:415-427.11. Faulkner JA, Brooks SV, Opiteck JA. Injury to skeletal muscle fibres duringcontractions: conditions of occurrence and prevention. Physical Therapy 1993 ;73( 12):91 1-921.146APPENDIX FOURRadio-isotope Investigation of Acute InflammationI Labelling procedureFifty millilitres (ml) of blood was taken by venipuncture from each of the subjects.In the lab the blood was separated into two tubes. One tube of 10 ml was centrifuged at3000 revolutions/minute (rpm) for 20 minutes, while a surfactant was added to the rest ofthe blood and it was allowed to separate for 45 minutes. Platelet poor plasma (PPP) wasremoved from the 10 ml of blood, to be used in later stages. After 45 minutes leukocyterich plasma (LRP) was removed from the sedimented red blood cells and centrifuged at 900rpm for 10 minutes. After 10 minutes all of the plasma was removed from the leukocyte,or white blood cell (WBC), pellet. One ml of PPP was added to the pellet and the cellswere re-suspended.Next the Technetium-99m (Tc-99m) HMPAO labelling solution was prepared. Theextract of Tc-99m was not more than four hours old. To a vial of HMPAO, 925megabequerels (MBq) of Tc-99m was aseptically added. To make up a total volume of fiveml, low 02 saline was added. This mixture was allowed to incubate for three minutes atroom temperature. Following incubation, the next step was a liquid extraction qualitycontrol procedure to determine the percentage of HMPAO. Not more than 20% impuritiesor less than 80% HMPAO was found. From the mixture four ml (740 MBq) was drawnfor the WBC labelling procedure.The four ml Tc-99m HMPAO was added to the WBC, the cells were re-suspended,and incubated for 20 minutes. Following incubation, the cells were spun at 800 rpm for147ten minutes. Then the supernatant was removed, which contained the Tc-99m not labelledto the WBC. Following the removal of the supematant, the Tc-99m labelled WBC were resuspended with three ml of PPP.The subject dose was drawn and a second quality control procedure wasperformed. In the dose calibrator, the activity of the supernatant and the cells wasmeasured to obtain a labelling efficiency. Labelling efficiency is usually between 50 and60%. The trypan blue exclusion test was done to determine the number of dead cells. Lessthan five dead cells should be seen in the field of view under the microscope. The dosagesdrawn for the subjects ranged from 235 - 454 MBq.II Analysis of the Presence of Tc-99m WBCRegions of Interest (ROl)After the series of four scans was completed, computer analysis of regions of interest(ROl) was used to determine the count/pixel of gamma radiation. Four areas of thequadriceps muscle were chosen as the ROT - the antero-distal aspect from the lateral view,the anterior aspect from the lateral view, the medial aspect from the anterior view and thelateral aspect from the anterior view (Figure 4).The analyses of the ROl were undertaken on three separate occasions by twoscorers. The ROl were carefully drawn to avoid the femur and the femoral circulation.Figure 20 is an example of the mean count/pixel of the three analyses as well as thestandard deviation (SD) for one subject for the antero-distal ROl. The figure illustrates thedifferences in the presence of Tc-99m WBC between the two legs, and the decay of the Tc99m over time. For all subjects the exercise leg was the right leg and the non-exercise legthe left leg.148Figure 20. Subject 4: Antero-IDistal Region of Interest. Values are means ± SD. See textfor explanation.149Figure 203o- nI’25 —-4-— Exercise Leg• Non-exercise Leg-J— r.—2OciI—zI15—CL_) Iz-510152025TIME (Hours)150The SD is a useful measure of variation within a given set of data. However, ifthere are large differences in the means within the data set a measure of relative variationallows comparisons to be made. The coefficient of variation (CV) expresses the SD as apercentage of the mean (SD/mean x 100) (1). Thus, the CV served as a measure of inter-observer variability in this study. Table 16 is a summary of the mean count/pixel, as wellas the SD, of each ROT at each test time for both legs for all of the subjects. Table 17 is asummary of the CV of each ROT at each test time for both legs for all of the subjects.From Table 17 it can be seen that most of the variability occurred in the medial andlateral aspects as these were the ROT that were the most difficult to draw from an anatomicalperspective. As well, the variability between observers increased at the later time periodsas the scans were less well defined due to the decaying Tc-99m. The ROT with the leastinter-observer variability was the anterior ROI.CalculationsFirst the ROT of the non-exercise leg was subtracted from the ROl of the exerciseleg to eliminate the background levels of Tc-99m. Table 18 summarizes the subtraction ofthe background for each ROT for each subject.Next, the physical decay of the Tc-99m was corrected using a decay table. Thecalibration time, or the time at which the subject dose is drawn, is considered hour 0 and isthe time at which 100% of the Tc-99m is present. From calibration time to the scan timesof two, four, 20 and 24 hours were three, five, 21 and 25 hours respectively. The physicaldecay, or the fraction of the Tc-99m remaining, at those times were 0.708, 0.562, 0.089,and 0.056 respectively. The mean count/pixel of each ROT of the exercise leg, with the151TABLE 16MEAN COUNT/PIXEL (± 1 SD) FOR EACH REGION OF INTERESTFOR EACH SUBJECT AT EACH TEST TIMEANTERO-DISTALSubject 2 Hours 4 Hours 20 Hours 24 Hours1 Right 15.56(0.3) 11.36(0.3) 2.63(0.5) 2.10(0.4)Left 10.43(0.2) 10.00(0.2) 2.50(0.4) 1.72(0.2)2 Right 14.73(0.7) 11.47(1.3) 3.19(0.2) 1.94(0. 1)Left 6.99(0.2) 5.38(0. 1) 2.09(0.4) 1.29(0. 1)3 Right 54.00(0.3) 24.21(1.8) 7.95(1.0) 3.48(0.4)Left 12.61(0.4) 7.20(0.4) 2.22(0.2) 1.55(0.1)4 Right 28.36(4.4) 21.03(0.9) 4.86(0.3) 3.37(0.2)Left 10.00(1.4) 9.51(0.3) 2.53(0.4) 1.93(0. 1)5 Right 24.40(2.1) 19.88(0.5) 4.97(0.3) 3.19(0.2)Left 13.17(0.8) 11.39(0.3) 3.23(0.1) 2.11(0.1)6 Right 13.55(1.0) 9.27(0.4) 2.99(0.4) 2. 13(0.3)Left 12.00(0.9) 7.60(0.5) 2.59(0.2) 1.75(0.4)7 Right 22.23(1.7) 19.71(0.2) 5. 17(0.5) 3.35(0.4)Left 13.78(0.5) 11.10(0. 1) 3.05(0.2) 2.42(0.5)8 Right 29.59(2.9) 25.33(1.9) 5.71(0. 1) 3.45(0. 1)Left 9.89(0.5) 7.96(0.2) 2.31(0. 1) 1.56(0.2)9 Right 67.55(4.2) 63.98(2.9) 10.52(0.9) 2.55(0.1)Left 12.22(0.5) 9.24(0.2) 2.49(0.3) 1.94(0.1)10 Right 123.33(2.5) 89.21(3.4) 19.83(1.8) 9.01(5.9)Left 14.78(1.1) 11.57(0.7) 2.90(0.3) 1.99(0. 1)152ANTERIORSubjects 2 Hours 4 Hours 20 Hours 24 Hours1 Right 15.95(0.7) 11.96(0.7) 2.76(0.1) 2.15(0.1)Left 13.97(0.8) 10.74(0.4) 2.70(0. 1) 2.07(0. 1)2 Right 12.62(0.8) 9.00(0.2) 2.63(0.3) 1.96(0.1)Left 9.60(0.7) 8.00(0.7) 2.18(0.1) 1.60(0.3)3 Right 20.81(0.7) 12.96(0.2) 4.11(0.1) 2.71(0.1)Left 13.49(1.1) 9.87(0.6) 2.69(0. 1) 2.09(0.1)4 Right 15.65(0.7) 14.74(0.9) 3.45(0.1) 2.39(0.1)Left 12.54(0.6) 9.43(0.8) 2.59(0. 1) 1.84(0.1)5 Right 16.77(1.7) 13.91(0.8) 3.66(0.1) 2.80(0.1)Left 14.60(1.2) 12.57(0.6) 3.47(0. 1) 1.84(0.6)6 Right 16.23(1.0) 10.58(0.6) 4. 10(0.4) 3.02(0. 1)Left 13.09(1.2) 9.71(0.3) 3.48(0.3) 2.23(0.2)7 Right 20.92(1.5) 17.99(1.49) 6.39(0.4) 4.36(0.1)Left 16.49(1.1) 13.12(1.1) 3.46(0.1) 3.05(0.2)8 Right 13.77(0.7) 11.06(0.5) 2.85(0.1) 1.95(0.1)Left 10.32(0.6) 9.41(0.6) 2.62(0.1) 1.82(0.1)9 Right 22.24(0.9) 17.04(0.6) 4.53(0.2) 3.05(0. 1)Left 14.07(1.2) 11.26(0.8) 2.89(0.1) 2.28(1.1)10 Right 22.99(1.6) 21.98(0.6) 6.53(0.5) 5.36(0.7)Left 17.38(1.7) 13.80(0.6) 2.86(0.1) 2. 16(0.2)153MEDIALSubjects 2 Hours 4 Hours 20 Hours 24 Hours1 Right 20.70(2.8) 13.73(1.0) 3.21(0.4) 2.54(0.1)Left 19.48(2.6) 10.51(0.5) 2.83(0.2) 1.92(0.2)2 Right 11.11(2.3) 10.35(1.1) 3.73(0.1) 2.13(0.2)Left 8.25(1.5) 6.68(0.3) 2.04(0.3) 1.60(0.2)3 Right 33.31(3.0) 18.19(5.6) 6.42(0.5) 3.96(0.2)Left 20.43(2.6) 10.87(0.6) 2.48(0.1) 2.16(0.3)4 Right 18.32(1.6) 20.64(1.4) 3.91(0.3) 2.84(0.5)Left 13.77(2.1) 12.45(1.1) 2.84(0.2) 2.02(0.1)5 Right 21.86(2.9) 12.85(2.9) 3.84(0.4) 3.07(0.7)Left 21.21(3.6) 11.51(3.7) 3.28(0.5) 2.15(0.1)6 Right 17.61(3.2) 10.98(2.2) 4.05(1.3) 2.87(0.7)Left 15.54(1.2) 11.54(2.5) 3.45(0.3) 2.14(0.1)7 Right 19.94(2.7) 14.91(3.6) 5.80(2.1) 3.53(1.4)Left 18.31(0.8) 13.41(0.1) 3.44(0.5) 2.08(0.3)8 Right 13.49(0.4) 11.92(1.0) 2.73(0.3) 1.85(0.2)Left 11.29(0.4) 10.62(0.6) 2.64(0.1) 1.61(0.1)9 Right 26.13(8.3) 22.58(7.4) 6.24(3.2) 3.77(1.1)Left 17.59(1.7) 12.78(0.7) 3.01(0.1) 2.24(0.1)10 Right 32.62(3.0) 26.03(8.5) 5.86(0.7) 3.71(0.7)Left 19.57(1.5) 13.63(1.7) 3.35(0.2) 2.36(0.1)LATERALSubjects 2 Hours 4Hours 20 Hours 24 Hours1 Right 18.59(1.0) 12.72(1.2) 2.64(0.2) 2.29(0.2)Left 19.25(2.1) 13.50(1.0) 2.49(0.5) 2.11(0.2)2 Right 13.82(0.7) 10.60(0.9) 3.01(0.5) 1.96(0.3)Left 11.42(0.4) 9.18(0.5) 2.30(0.1) 1.61(0.1)3 Right 27.29(1.8) 16.89(1.5) 5.21(0.7) 2.98(0.3)Left 20.63(1.3) 11.60(1.8) 2.71(0.4) 2.05(0.2)4 Right 25.00(7.8) 15.93(1.9) 3.71(0.4) 2.64(0.2)Left 16.69(1.3) 12.25(1.2) 2.64(0.3) 2.14(0.1)5 Right 27.76(7.5) 17.98(4.6) 4.39(0.9) 3.09(0.7)Left 2 1.43(2.5) 14.28(2.0) 3.80(0.1) 2.66(0.2)6 Right 17.47(0.6) 11.79(1.4) 4.04(0.6) 2.55(0.5)Left 16.15(0.7) 12.52(1.0) 4.10(0.4) 3.02(0.1)7 Right 20.84(1.2) 17.68(1.3) 6.28(0.5) 3.78(0.4)Left 19.66(2.0) 13.89(2.3) 3.43(0.4) 2.64(0.3)8 Right 13.67(1.5) 12.88(0.3) 2.88(0. 1) 2.00(0.2)Left 12.47(0.7) 10.55(1.1) 2.45(0.3) 1.74(0.1)9 Right 25.58(0.5) 21.13(3.0) 4.95(0.5) 3.13(0.2)Left 19.87(1.5) 13.59(0.4) 3.77(0.1) 2.36(0.1)10 Right 29.58(4.6) 21.34(5.0) 4.66(0.5) 3.21(0.8)Left 18.35(5.5) 14.74(4.2) 3.35(0.5) 2.23(0.4)TABLE17COEFFICIENTOFVARIATION(%)FORTHEREGIONSOFINTERESTSubjectAntero-DistalAnteriorMedialLateralHourRLRLRLRL21.931.924.395.7313.5313.355.3810.9142.642.005.853.727.284.769.437.402019.0116.063.623.7012.467.077.5820.002419.0511.634.654.813.9410.428.739.48224.752.866.347.2920.7018.185.073.50411.331.862.228.7510.634.498.495.45206.2719.1411.414.592.6814.7116.614.35245.157.755.1018.759.3912.5015.316.213210.373.173.368.159.0012.736.606.3047.435.551.546.0830.795.528.8815.522012.589.012.433.727.794.0313.4414.762414.376.453.694.785.0513.8910.079.764215.5114.004.474.788.7315.2531.207.7944.763.166.788.486.788.8411.939.80206.1715.812.901.167.677.0410.7811.36245.932.071.674.8917.611.987.584.67U,528.616.0710.148.2213.2716.9727.0211.6742.522.635.754.7722.5732.1525.5814.01206.043.102.730.5810.4215.2420.501.32246.272.371.7932.6122.804.1922.657.52627.387.506.169.1718.177.723.434.3344.316.585.673.0920.0321.6611.877.992013.387.729.768.6232.108.7014.8515.342414.0822.863.318.9724.394.6719.6113.57727.653.637.176.6713.544.375.7610.1741.020.908.348.3824.140.757.3514.40209.676.566.262.8936.2114.537.9611.662411.9420.662.296.5639.6614.4210.5811.36829.805.065.075.812.973.5410.975.6147.902.514.526.388.395.652.3310.43201.754.333.513.8210.993.793.4712.24242.9012.825.135.4910.814.6710.005.75926.224.094.058.5331.769.661.957.5444.532.163.527.1032.775.4814.202.94208.5612.054.423.4751.283.3210.102.65243.925.153.284.3929.184.466.394.24ci,ci-’1022.037.436.969.789.207.6615.5529.9743.806.052.734.3532.6512.4723.4328.49209.0810.347.663.5011.955.9710.7314.932465.565.0313.069.2618.874.2424.9217.94ITABLE 18SUBTRACTION OF THE BACKGROUND(Exercise Leg - Non- exercise Leg)Subjects Hour Antero-Distal Anterior Medial Lateral2 5.14 1.98 1.22 -0.664 1.36 1.22 3.22 -0.7820 0.13 0.06 0.38 0.1424 0.38 0.08 0.63 0.182 2 7.74 3.02 2.86 2.404 6.10 1.00 3.66 1.4220 1.10 0.48 1.69 0.7024 0.65 0.36 0.53 0.353 2 41.40 7.31 12.88 6.664 17.00 3.09 7.32 5.2820 5.73 1.42 3.94 2.5024 1.94 0.62 1.80 0.934 2 18.36 3.11 4.55 8.314 11.52 5.31 8.18 3.6820 2.33 0.86 1.07 1.0724 1.45 0.55 0.82 0.505 2 11.23 2.17 0.65 6.324 8.48 1.34 1.34 3.7020 1.74 0.18 0.56 0.5824 1.08 0.96 0.93 0.436 2 1.55 3.14 2.08 1.324 1.67 0.88 -0.56 -0.7320 0.41 0.63 0.60 0.7824 0.37 0.78 0.35 0.737 2 8.45 4.43 1.63 1.184 8.61 4.87 1.51 3.7920 2.11 2.92 2.37 2.8524 0.93 1.31 1.45 1.148 2 19.70 3.46 2.20 1.204 17.38 1.65 1.29 2.3320 3.41 0.23 0.09 0.4324 1.89 0.13 0.25 0.261579 2 55.34 8.17 8.54 5.724 54.74 5.78 9.80 7.5420 8.03 1.66 3.23 1.1824 0.61 0.76 1.53 0.7610 2 108.55 5.61 13.05 11.234 77.64 8.18 12.40 6.6020 16.93 3.67 2.51 1.3124 7.01 3.20 1.35 0.98158159background subtracted, was divided by the fraction of Tc-99m remaining to arrive at thedecay corrected values for each subject and each ROT (Figures 21 - 25). The figures forsubjects 1 and 6 have a different Y scale to the others because they both had negativevalues. The Y scales for Figure 25 are also different because the presence of Tc-99m WBCin at least one of the ROT was much greater than for the other subjects.Finally, because the dosages of Tc-99m WBC varied between subjects, the datawere normalized to the peak response of each subject for each ROT. The non-normalizedand normalized data are presented in Tables 19 and 20 respectively.III Possible Mechanisms Contributing to the Responses of TwoSubjectsThe decay corrected differences for each ROT for two subjects - subject 6 andsubject 10- represent the extreme responses of this data set (Figures 23 and 25, Table 19).The presence of Tc-99m WBC in the antero-distal ROT for subject 10 ranged from 18 timesgreater at 24 hours post-exercise to 70 times greater at two hours post-exercise compared tothe responses for subject 6. The magnitude of different responses in the presence of Tc99m WBC were not as great in the other ROT but still they ranged from 1.68 times greaterto 20 times greater numbers of Tc-99m WBC throughout the quadriceps muscle for subject10 versus subject 6.One of the major differences in the responses of the two subjects was that thepresence of Tc-99m WBC in the quadriceps muscle was evident immediately at two hourspost-exercise in subject 10 (Figure 25), while the responses were low in subject 6 until 20hours post-exercise (Figure 23). The second major difference was that the presence of Tc99m WBC in the antero-distal ROT of subject 10 was substantially greater than the other160The Presence of Technetium-99m White Blood Cells in the Four Regions of Interest forEach Subject. The background has been subtracted and the counts/pixel decay-corrected. The legends on each figure represent the four regions of interest.Figure 21. Subject 1. The scale on the y axis is the same as Subject 6, but different to theother subjects, because Subjects 1 and 6 both had negative values.Subject 2.Figure 22. Subject 3 and Subject 4.Figure 23. Subject 5 and Subject 6. The scale on the y axis for Subject 6 is the same asfor Subject 1.Figure 24. Subject 7 and Subject 8.Figure 25. Subject 9 and Subject 10. The scale on the y axis is different for these twosubjects from the other subjects because the presence of Tc-99m WBC in at leastone ROT was greater than for the other subjects..161SUBJECT 1Figure 217051.5-33—14.5-—-•-- Medial• LateralAnterior-- Antero-Distal._••_ x-JLuazCizw-JLu‘C0I—zD0(-3w— I—0 5 10 15 20 25TIME (Hours)SUBJECT 2-4706050403020100-. :t ----Medial ±• Lateral—•°--- Anteriort- -i-- -X-- Antero-Distal—t--+Ii: ±I-I--I- -1—— * —— * t. I_)E--X - -0 5 10 15 20 25TIME (Hours)Figure 2270 -60 -SUBJECT 3162—--— MedialLateral— <>— Anterior- -X-- Antero-Distal50 -40 -3020 -10 -x4——_...,.\4———4——4.-—— ——-JUiC)C)Lii-JLii>caI—zC)C)zLii0 5 10 15 20 25TIME (Hours)SUBJECT 4070605040302010——I I—I+ —-—MediaI - -• LateralE---<>—- Anterior- -00 5 10 15 20 25TIME (Hours)-JUiaI—z0Liz4UI-JUIaIz0ciz4UIzFigure 2370SUBJECT 516360 -50 -403020-10-0—--— Medial• Lateral—°--- Anterior- -X-- Antero-Distalx>6- - --ew—7051.53314.5-40 5 10 15 20 25TIME (Hours)SUBJECT 6.--i‘ “Medial II—-S Lateral--4--— °— Anterior-- -X-- Antero-Distal-e-’..--•--H0 5 10 15 20 25TIME (Hours)-J‘CUI—z0C-)zLii-JLii‘CUI0C-)zLiiEFigure 24SUBJECT 71647060 T40+I—30I201-4—I——--— Medial• Lateral‘—- Anterior- -X-- Antero-Distal-I-±-II-Ix—.1007060504030201000 5 10 15 20 25TIME (Hours)SUBJECT 8—“—MediaI±Lateral---- a---- Anterior—- -X-- Antero-Distal±F-F!.-----i: -x-------F- XrIII—L‘—-—— —. —— —(:— —0 5 10 15TIME (Hours)20 25165--•-- MedialFigure 25_____— LateralSUBJECT 9“ Anterior100 I- —X-- Antero-Distal/- - --80— x>cJ’JI—zDL)z 40___-.-—I_u20—-0-. I I I -0 5 10 15 20 25TIME (Hours)— MedialLateralSUBJECT 1 0 Anterior200-- -X-- Antero-Distal--y’1501--100—cizi_u50.,-—--—-o- I-0 5 10 15 20 25TIME (Hours)166TABLE 19TECHNETIUM-99M WHITE BLOOD CELLS FOR EACH REGION OF INTEREST ATEACH TEST TIME(Mean Count/Pixel)ANTERO-DISTALSubject 2 Hours 4 Hours 20 Hours 24 Hours1 7.25 2.42 1.51 6.732 10.93 10.84 12.39 11.603 58.47 30.25 64.39 34.574 25.94 20.50 26.21 25.825 15.86 15.09 19.59 19.286 2.19 2.97 4.56 6.687 11.94 15.32 23.76 16.678 27.83 30.92 38.27 33.759 78.15 97.41 90.22 10.8910 153.31 138.16 190.25 125.25ANTERIORSubjects 2 Hours 4 Hours 20 Hours 24 Hours1 2.80 2.17 0.64 1.432 4.26 1.77 5.02 6.433 10.33 5.50 15.96 11.134 4.39 9.44 9.70 9.825 3.06 2.40 2.07 17.146 4.44 1.56 7.03 14.007 6.26 8.66 32.85 23.458 4.89 2.94 2.59 2.389 11.53 10.29 18.60 13.6410 7.93 14.56 41.27 57.20167MEDIALSubjects 2 Hours 4 Hours 20 Hours 24 Hours1 1.72 5.74 4.27 11.182 4.04 6.52 18.99 9.413 18.20 13.02 44.34 32.094 6.43 14.56 12.02 14.645 0.92 2.39 6.33 16.546 2.91 -1.00 6.73 13.037 2.29 2.68 26.58 25.898 3.11 2.30 0.98 4.399 12.07 17.43 36.30 27.2710 18.43 22.07 28.20 24.11LATERALSubjects 2 Hours 4Hours 20 Hours 24 Hours1 -0.94 -1.39 1.62 3.162 3.39 2.54 7.91 6.253 9.41 9.40 28.06 16.614 11.74 6.55 12.06 8.985 8.93 6.58 6.56 7.636 1.87 -1.30 8.76 6.187 1.67 6.73 31.99 20.318 1.69 4.16 4.86 4.649 8.07 13.41 13.22 13.6410 15.87 11.74 14.75 17.50168TABLE 20NORMALIZED DATA FOR EACH REGION OF INTEREST(%)ANTERO-DISTALSubject 2 Hours 4 Hours 20 Hours 24 Hours1 100.00 33.38 20.83 92.832 88.22 87.49 100.00 93.623 90.81 46.98 100.00 53.694 98.97 78.21 100.00 98.515 80.96 77.03 100.00 98.426 32.78 44.46 68.26 100.007 50.25 64.48 100.00 70.168 72.72 80.79 100.00 88.199 80.23 100.00 92.31 11.1810 80.58 72.62 100.00 65.83ANTERIORSubjects 2 Hours 4 Hours 20 Hours 24 Hours1 100.00 77.50 22.86 51.072 66.25 27.53 78.07 100.003 64.72 34.46 100.00 69.744 44.70 96.13 98.78 100.005 17.84 13.99 12.07 100.006 31.71 11.14 50.21 100.007 19.06 26.36 100.00 71.398 100.00 60.12 52.97 48.679 61.99 55.32 100.00 73.3310 13.86 25.45 72.15 100.00169DIALSubjects 2 Hours 4 Hours 20 Hours 24 Hours1 15.38 51.34 38.19 100.002 21.27 34.33 100.00 49.553 41.06 29.36 100.00 72.374 43.92 99.45 82.10 100.005 05.56 14.45 38.27 100.006 22.33 -7.67 51.65 100.007 08.62 10.08 100.00 97.408 70.84 52.39 22.32 100.009 33.25 48.02 100.00 75.1210 65.35 78.26 100.00 85.50LATERALSubjects 2 Hours 4Hours 20 Hours 24 Hours1 -29.75 -43.99 51.27 100.002 42.86 32.11 100.00 79.013 33.54 33.50 100.00 59.194 97.35 54.31 100.00 74.465 100.00 73.68 73.46 85.446 21.35 -14.84 100.00 70.557 05.22 21.04 100.00 63.498 34.77 85.60 100.00 95.479 59.24 98.31 96.92 100.0010 90.69 67.09 84.29 100.00170ROT for this subject (Figure 25), and was the greatest of any of the subjects (Table 19).The response also continued to be high over 24 hours. The presence of Tc-99m WBC inthe antero-distal ROI of subject 6 was less than the other ROT for this subject, except atfour hours post-exercise (Figure 23), and amongst the lowest responses of all of thesubjects (Table 19).As the dosages of Tc-99m WBC varied amongst the subjects, it might be suspectedthat subject 10 had a higher dosage than subject 6 but in fact the opposite was true. Thedosage prepared for subject 10 was 331 MBq and for subject 6 it was 454 MBq Tc-99mWBC. In both cases the Tc-99m WBC were injected within one hour of calibration. Thebackground levels and the physical decay of Tc-99m were accounted for in the datapreparation for both subjects, as previously described.Among the biological factors to be considered, the vascular response to theeccentric exercise protocol may have been different for the two subjects. Tt is not known ifthere was a difference between subjects in the blood flow to the muscle, which wouldnormally be increased following exercise and also as a result of acute inflammation (2). Ttis also typical to have an increase in vascular permeability in response to acute inflammation(2) which may have caused swelling and subsequently decreased ROM. Subject 10 had animmediate decrease in ROM at two hours post-exercise which did not recover until 72hours post-exercise, while ROM did not change for subject 6 until 20 hours post-exercise(Figure 26A).Subject 10 had an immediately elevated response in the presence of Tc-99m WBCin the exercised muscle, particularly in the antero-distal ROI (Figure 25), while subject 6had a gradual increase in the presence of Tc-99m WBC up to 20 and 24 hours post-exercise(Figure 23). The presence of greater numbers of WBC suggests that there was more171damage in the quadriceps muscle of subject 10 than subject 6. Evans and Cannon (3) havestated that the generation of an acute inflammatory response depends, at least to someextent, on the duration and on the intensity of exercise. Because the intensity was underthe control of the subject, this may have been one of the factors contributing to the differentresponses for these two subjects.Evans and Cannon (3) have stated that tissue damage or infection initiate a host ofdefense reactions that promotes clearance of the damaged tissue and sets the stage forrepair. Complement is the name given to a complex series of plasma proteins which whentriggered produces a rapid, highly amplified response to cell injury or invasion (4). Thecomplement system can be activated by damaged host cells via the alternative pathway (3).Some of the end products of the complement cascade are C3a and C5a (4) which areactivators of mast cells, neutrophils and monocytes (3). Following the vascular response,neutrophils are attracted to the area of tissue damage within minutes and they remainpresent in the tissue for up to 24 hours following the insult (3). Neutrophils are drawn tothe site of injury by chemoattractants such as C5a (3).Circulating monocytes derive into macrophages in the tissue (2). The lifespan ofmacrophages in the tissues varies from days to months (3). Neutrophils and macrophagesboth phagocytize, or clean up, the damaged tissue, however, in the process, neutrophilsmay also damage the host tissue (3). Macrophages, in addition to clearing the debris andthe neutrophils, begin the process of repair and are important in the regeneration andstabilization of the tissue (3).Figures 26 - 28 illustrate the responses of the functional measures of subject 6 andsubject 10 compared to the group mean ± one SD. There was an immediate response in allof the functional measures for subject 10 and only ROM and fatigue of vastus lateralis (VL)172The Responses of Two Subjects Compared to the Group Mean ± One Standard Deviation.Figure 26. A. Range of Motion of the Right Knee. B. Eccentric Torque.Figure 27. A. Intensity of Soreness. B. Unpleasantness.Figure 28. A. Vastus Lateralis Median Frequency. B. Vastus Medialis Median Frequency.Cm x 0 C 1 0cD r’Jm 0 C 00)-I,u:•>(D 0,ECCENTRICTORQUE(Nm)NJ-CT0)0N)-0000000-o-‘ CD 0 NiRANGEOFMOTION(degrees)-o CD--N)Ni(.*JWoC-n0(‘10(rI....,-_..TI....j-.--..-\ ‘I‘I‘If4IMEANSCOREMEANSCORE-J U,o0U,0(11-DDZ.OO--Jo_)r’o(I,z rn U,(I)H m 0 U,r\j 0 F’) i’)(t Ni Ni 0 coI I IH 0 C (I,I--ou’Cc-nC(‘1-rf-i.--I——————————4--.00I, II.SLOPEOFTHEMEDIANFREQUENCYIIIIppppp -f_r._r-..-r--IT .4SNH,ISLOPEOFTHEMEDIANFREQUENCYLiIIIpp0ro-b—(A)p C C,(t ri-o—I (tI0 r0 U)‘-I,-S (D ODOD0.1,,0 (I)OD\I________II— —40,176had recovered by 72 hours post-exercise. Conversely, intensity of soreness,unpleasantness and ROM did not increase noticeably until 20 hours post-exercise forsubject 6 and had recovered by 72 hours post-exercise. Eccentric torque had recovered tolevels greater than the pre-test by four hours, and although it declined again at 24 hourspost-exercise, the torque at 20, 48 and 72 hours post-exercise was also greater than the pretest. Slight fatigue was evident only at two hours post-exercise in both VL and VM forsubject 6.When the differences in the functional responses of the two subjects are consideredin light of the substantial differences in the presence of Tc-99m WBC in the antero-distalROT, the results suggest that the presence of Tc-99m WBC, and thus the extent of damage,in the distal segment of the quadriceps muscle may have been the most important factorcontributing to the subjects’ different functional responses. The location of the anterodistal ROT in this study is in the region of the musculo-tendinous junction of the quadricepsmuscle. Stauber (5) has suggested that connective tissue damage is a component ofexercise-induced muscle injury. However, there has been little study in this area and moredirect evidence of connective tissue damage is needed.Friden and colleagues (6) reported more pronounced Z line disorganization inhuman muscle three days after an eccentric exercise protocol than at one hour following theexercise, suggesting a progression of the tissue damage. Fritz and Stauber (7) reporteddegradation of proteoglycan, a component of extracellular matrix (ECM), in the areasurrounding damaged myofibres of rats 24- 72 hours post-injury. The progression ofdamage in the muscle may be related to the activity of the WBC, especially neutrophils, atthe site of injury. Neutrophils have little ability to differentiate between foreign and hostcells (8). They also release an assortment of oxygen-dependent and oxygen-independentproducts that are capable of destroying normal cells and dissolving connective tissue (8).177Elastase and collagenase are two proteolytic enzymes that have the greatest potential fortissue damage (8). These enzymes attack the ECM, presumably to aid in migration of theneutrophils during the inflammatory response (9). As well, the neutrophil’s secretory andinjurious potential at the site of injury may be amplified by priming agents from monocytesand macrophages or platelet activating factor (PAF) or tumor necrosis factor (TNF) (9).However, for healthy repair of tissue there must be a balance between tissue injury andprotection of the tissue. In addition to antioxidants within the tissue, there are physicalprocesses which are thought to be protective. Neutrophil migration into tissue ceases veryearly in the acute inflammation stage, chemoattractant activity may decrease, andneutrophils, both disintegrated and senescent, are actively removed by macrophages (9). Itis not known how neutrophils and macrophages interact in damaged muscle but theprogressive injury reported by others and the evidence of WBC within the muscle over 24hours from this study suggest that this is an area of further study.In summary, further research would benefit from collection of Tc-99m WBC dataover a longer period of time, such as 48 hours. This would require a second dosage of Tc99m WBC or use of another radio-isotope, such as Indium, which has a longer half life.Specific labelling of neutrophils and/or monocytes would allow specific description of theacute phase cellular response following eccentric exercise. More specific investigation ofthe musculo-tendinous junction following eccentric exercise may provide furtherinformation regarding the status of the connective tissue. And finally, if it were possible tokeep the dosages of Tc-99m WBC constant then correlations with functional measurescould be made which would also provide information about the importance of the locationof the muscle injury.178References1. Dame! WW. Biostatistics: A Foundation for Analysis in Health Sciences. (5th ecL)New York: John Wiley and Sons, 19912. Kent TH, Hart MN. Injury, Inflammation and Repair.Norwalk, CT: Appleton andLange, 19933. Evans WJ, Cannon JG. The metabolic effects of exercise-induced muscledamage.Baltimore: Williams and Wilkins, 1991:99- 125. Exercise and Sports ScienceReviews4. Roitt I. Essential Immunology. (7 ed.) London: Blackwell Scientific Publications,19915. Stauber WT, Clarkson PM, Fritz VK, Evans WJ. Extracellular matrix disruptionand pain after eccentric muscle action. Journal of Applied Physiology 1990;69(3):868-874.6. Fridén J, Sjostrom M, Ekblom B. Myofibrillar damage following intense eccentricexercise in man. International Journal of Sports Medicine 1983 ;4: 170-176.7. Fritz VK, Stauber WT. Characterization of muscles injured by forced lengthening.II. Proteoglycans. Medicine and Science in Sports and Exercise 1988;20(4):354-361.8. Weiss SJ. Tissue destruction by neutrophils. New England Journal of Medicine1989320(6) :365-375.9. Haslett C, Savill JS, Meagher L. The neutrophil. Current Opinion in Immunology1989;2: 10-18.179180APPENDIX FIVEIndividual Subject Data - Chapter One= missing dataSubject 1Age 42Gender FWeight (Kg) 68. 1Leg Tested RPhysical Activity (Hr/wk) 3Exercise Duration Shorter (180 reps)Test Session (Hour) pre-test 24 48 96 168DDS Soreness -10 8.32 14.68 9.32 8.73DDS Unpleasant. -10 0.77 2.53 2.82 0.61VAS * * * * *Ecc. Torque (N.m) 124 77 84 85 95CPK (lUlL) 55 397 340 455 3034OHP (umol/L) 39 33 63 51 62Cr(umol/L) 3034 3507 4175 3969 3918OHPICr 12.8 9.4 15.0 12.8 15.8Exercise Duration Longer (300 reps)Test Session (Hour) pre-test 24 48 96 168DDS Soreness -10 3.86 14.21 9.49 -6.91DDS Unpleasant. -10 -5.71 0.82 0.52 -8.32VAS * * * * *Eec. Torque (N.m) 125 109 98 113 113CPK (lUlL) 48 240 136 458 40801-P (umol/L) 93 86 88 51 90Cr (umollL) 4466 6435 6218 5061 646001-P/Cr 20.8 13.4 14.1 10.1 13.9181Subject 2Age 42Gender FWeight (Kg) 71.7Leg Tested RPhysical Activity (Hr/wk) 0Exercise Duration Shorter (180 reps)Test Session (Hour) pre-test 24 48 96 168DDS Soreness -10 -2.24 1.11 -7.23 -9.00DDS Unpleasant. -10 -9.33 -1.15 -8.46 -9.00VAS * * * * *Ecc. Torque (N.m) 108 92 101 118 126CPK (lUlL) 77 103 76 60 94OHP (umollL) 93 77 82 94 160Cr (umollL) 6224 6608 5828 6795 7424OHPICr 14.9 11.7 14.1 13.8 21.6Exercise Duration Longer (300 reps)Test Session (Hour) pre-test 24 48 96 168DDS Soreness -10 10.05 5.93 2.71 -10DDS Unpleasant. -10 0.32 4.86 -2.36 -10VAS 0.0 4.3 5.9 1.4 0.0Eec. Torque(N.m) 114 68 65 82 110CPK (lUlL) 35 99 86 95 97Ol-IP (umollL) 137 53 59 68 48Cr(umol/L) 4381 5410 5035 7199 6613OHP/Cr 31.3 9.8 11.7 9.5 7.3182Subject 3Age 20Gender FWeight (Kg) 55.8Leg Tested LPhysical Activity (Hr/wk) 3Exercise Duration Shorter (180 reps)Test Session (Hour) pre-test 24 48 96 168DDS Soreness -3.79 15.61 16.82 14.39 -10DDS Unpleasant. -10 6.82 6.52 -6.67 -10VAS 0.0 7.8 5.0 2.9 0.0Ecc. Torque (N.m) 138 84 99 113 124CPK (lUlL) 58 216 148 250 *01-P (umol/L) 139 524 302 240 255Cr(umol/L) 7707 17668 12713 13586 1097801-P/Cr 18.0 29.7 23.8 17.7 23.2Exercise Duration Longer (300 reps)Test Session (Hour) pre-test 24 48 96 168DDS Soreness -1.05 14.99 14.23 6.12 -10DDS Unpleasant. -10 0.77 3.73 -3.76 -10VAS * * * * *Eec. Torque (N.m) 144 80 96 124 125CPK (lUlL) 60 311 131 123 5901-P (umol/L) 244 174 367 225 338Cr(umol/L) 12519 9705 9609 8120 1051901-P/Cr 19.5 17.9 38.2 27.7 32.1183Subject 4Age 24Gender FWeight (Kg) 49.9Leg Tested RPhysical Activity (Hr/wk) 1Exercise Duration Shorter (180 reps)Test Session (Hour) pre-test 24 48 96 168DDS Soreness -9 -7.14 -6.52 -4.67 -8.20DDS Unpleasant. -9 -6.99 -8.09 -7.99 -8.94VAS 0.0 0.6 1.3 0.4 0.4Ecc. Torque (N.m) 43 91 70 66 57CPK (lUlL) 44 52 49 57 4101-P (umol/L) 349 344 191 204 245Cr(umollL) 11059 13759 12425 9457 13737OHPICr 31.6 25.0 15.4 21.6 17.8Exercise Duration Longer (300 reps)Test Session (Hour) pre-test 24 48 96 168DDS Soreness -9.18 10.5 11.44 14.44 -8.32DDSUnpleasant. -10 10.17 10.36 12.52 -10VAS 0.2 8.6 7.3 5.5 0.0Ecc. Torque (N.m) 58 39 87 70 76CPK (lU/L) 67 132 78 44 3201-P (umol/L) 357 250 190 353 148Cr(umol/L) 11292 11162 8263 12336 706001-P/Cr 31.6 22.4 22.9 28.6 20.9184Subject 5Age 48Gender FWeight (Kg) 56.8Leg Tested RPhysical Activity (Hrlwk) 0Exercise Duration Shorter (180 reps)Test Session (Hour) pre-test 24 48 96 168DDS Soreness -5.71 13.83 11.64 7.27 -7.49DDS Unpleasant. -9.71 -0.29 -1.89 -7.65 -9.14VAS 0.2 2.3 1.6 2.3 1.4Ecc. Torque (N.m) 108 80 94 97 113CPK (lUlL) 72 176 135 106 106OHP (umollL) * 46 40 44 47Cr(umol/L) * 4987 4157 3259 5163OHP/Cr * 9.2 9.6 13.5 9.1Exercise Duration Longer (300 reps)Test Session (Hour) pre-test 24 48 96 168DDS Soreness -4.17 10.91 14.21 -4.02 -9.18DDS Unpleasant. -9.14 -4.49 -3.99 -8.71 -10VAS 0.0 2.5 4.6 0.9 0.9Eec. Torque (N.m) 66 55 62 63 79CPK (lU/L) 83 158 113 81 123OHP (umol/L) 65 94 43 48 46Cr (umol/L) 8445 5327 4948 4160 4838OI-IP/Cr 7.7 17.65 8.7 11.54 9.51185Subject 6Age 27Gender MWeight (Kg) 70.4Leg Tested RPhysical Activity (Hr/wk) 4Exercise Duration Shorter (180 reps)Test Session (Hour) pre-test 24 48 96 168DDS Soreness -8.77 9.86 11.91 12.53 8.21DDS Unpleasant. -9.38 7.86 10.58 3.47 -5.67VAS 0.2 5.5 7.3 6.8 2.3Eec. Torque (N.m) 177 119 85 118 122CPK (lU/L) 154 324 281 340 726OHP (umol/L) 174 362 355 341 225Cr(umol/L) 14531 20175 20965 20187 19835OHP/Cr 19.7 17.9 16.9 16.9 11.3Exercise Duration Longer (300 reps)Test Session (Hour) pre-test 24 48 96 168DDS Soreness -7.52 14.38 13.97 * -9.09DDS Unpleasant. -10 6.61 9.36 * -10VAS 0.0 5.0 6.6 * 0.0Ecc. Torque (N.m) 165 104 100 * 168CPK (lUlL) 121 208 180 * 1257Ol-IP (umol/L) 100 190 247 * 40Cr(umollL) 12608 16327 20064 * 10332OHPICr 7.9 11.6 12.3 * 39186Subject 7Age 21Gender FWeight (Kg) 59Leg Tested RPhysical Activity (Hr/wk) 3Exercise Duration Shorter (180 reps)Test Session (Hour) pre-test 24 48 96 168DDS Soreness -10 10.70 9.61 -0.23 *DDS Unpleasant. -10 -3.02 -0.46 -10 *VAS 0.0 5.0 5.0 1.1 *Ecc. Torque (N.m) 146 88 76 93 *CPK (lUlL) * 140 * 48 *OI-IP(umol/L) 431 425 220 114 *Cr(umol/L) 9618 17981 6889 7432 *OHPICr 44.8 23.6 31.9 15.3 *Exercise Duration Longer (300 reps)Test Session (Hour) pre-test 24 48 96 168DDS Soreness -10 6.71 0.99 -10 -10DDS Unpleasant. -10 -5.29 -9.56 -10 -10VAS 0.0 2.3 0.1 0.0 0.0Ecc.Torque(N.m) 77 112 118 83 123CPK (lU/L) 34 131 * 53 88OHP(umol/L) 159 469 230 364 356Cr(umollL) 12560 19393 11126 14080 17152OHPICr 12.6 24.2 20.6 25.8 20.7187Subject 8Age 38Gender FWeight (Kg) 48.6Leg Tested RPhysical Activity (Hr/wk) 1Exercise Duration Shorter (180 reps)Test Session (Hour) pre-test 24 48 96 168DDS Soreness -10 1.53 13.32 -10 -10DDS Unpleasant. -10 -12.42 -5.52 -10 -10VAS 0.0 1.8 0.9 0.0 0.0Ecc.Torque(N.m) 118 84 87 117 126CPK(IU/L) * * * * *OHP(umollL) 167 75 106 151 79Cr (umol/L) 5681 6993 6410 5481 7476OHPICr 29.0 10.7 16.5 27.5 10.5Exercise Duration Longer (300 reps)Test Session (Hour) pre-test 24 48 96 168DDS Soreness -10 6.42 -0.73 4.14 -7.91DDS Unpleasant. -10 -9.47 -10 -9.43 -9.09VAS 0.0 6.4 1.4 1.1 0.0Eec. Torque (N.m) 74 75 104 101 106CPK (lUlL) 61 152 157 129 148OHP(umollL) 97 83 57 115 53Cr (umollL) 8926 8536 4641 5806 5806OHPICr 10.8 9.7 12.2 19.8 9.1188Subject9Age 23Gender FWeight (Kg) 62.2Leg Tested RPhysical Activity (Hr/wk) 2Exercise Duration Shorter (180 reps)Test Session (Hour) pre-test 24 48 96 168DDS Soreness 1.44 12.30 14.61 -4.79 -9.67DDS Unpleasant. -9.15 -2.00 1.11 -10 -10VAS 1.7 3.6 4.1 0.3 0.2Eec. Torque (N.m) 108 60 95 88 101CPK (lUlL) 52 147 80 60 53OHP(umollL) 67 112 296 179 113Cr(umol/L) 2723 5397 7877 8104 4410OHPICr 24.6 20.8 37.6 22.1 25.6Exercise Duration Longer (300 reps)Test Session (Hour) pre-test 24 48 96 168DDS Soreness -0.88 13.11 13.03 -10 -10DDS Unpleasant. -6.09 -0.85 2.38 -8.42 -10VAS 1.2 6.4 7.9 0.0 0.0Eec. Torque (N.m) 104 75 50 97 107CPK (lUlL) 63 192 196 1245 610OI-IP(umolIL) 164 145 126 214 301Cr(umol/L) 6453 7940 5748 9917 11781OHPICr 25.4 18.3 21.9 21.5 25.5189Subject 10Age 19Gender FWeight (Kg) 53.6Leg Tested RPhysical Activity (Hr/wk) 3Exercise Duration Shorter (180 reps)Test Session (Hour) pre-test 24 48 96 168DDS Soreness -8.26 5.20 9.56 -0.47 -9.76DDS Unpleasant. -8.70 -1.21 6.77 -3.35 -10VAS 1.6 5.7 5.9 2.7 0.1Eec. Torque(N.m) 57 99 81 100 119CPK (lUlL) 152 503 345 145 113OHP(umol/L) 147 227 370 110 286Cr (umol/L) 7225 9549 10679 6268 12575OHP/Cr 20.3 23.7 34.6 17.5 22.7Exercise Duration Longer (300 reps)Test Session (Hour) pre-test 24 48 96 168DDS Soreness -6.88 9.88 10.00 7.61 -4.86DDS Unpleasant. -9.65 6.39 10.24 8.62 -8.08VAS 0.7 7.7 9.6 5.9 0.9Eec. Torque (N.m) 110 102 * 81 105CPK (lU/L) 81 493 248 318 480OHP(umol/L) 91 72 165 200 101Cr (umol/L) 5337 6047 6338 8005 2993OI-IP/Cr 17.0 11.9 26.0 24.9 33.7190Subject 11Age 20Gender MWeight (Kg) 77.2Leg Tested RPhysical Activity (Hr/wk) 0Exercise Duration Shorter (180 reps)Test Session (Hour) pre-test 24 48 96 168DDS Soreness 3.59 5.15 14.49 0.05 -9.47DDS Unpleasant. -10 -5.23 0.77 -8.26 -10VAS 0.0 4.8 5.5 2.7 0.0Ecc.Torque(N.m) 180 144 156 208 221CPK (lUlL) 79 296 189 204 100OHP (umollL) 343 255 520 257 147Cr(umol/L) 17809 27571 23148 18532 18906OHPICr 19.3 9.3 22.5 13.9 7.8Exercise Duration Longer (300 reps)Test Session (Hour) pre-test 24 48 96 168DDS Soreness -7.06 13.64 11.38 15.24 -6.86DDS Unpleasant. -10 -3.15 13.26 4.08 -7.70VAS 0.5 5.6 8.4 5.8 1.4Ecc.Torque(N.m) 187 150 52 135 202CPK (lU/L) 88 396 2057 2545 369OHP(umol/L) 307 313 171 361 153Cr(umollL) 18188 27384 19449 17862 16516OHPICr 16.9 11.4 8.8 20.2 9.3191Subject 12Age 28Gender FWeight (Kg) 65.8Leg Tested RPhysical Activity (Hr/wk) 2Exercise Duration Shorter (180 reps)Test Session (Hour) pre-test 24 48 96 168DDS Soreness -9.21 14.06 14.30 8.62 -7.47DDS Unpleasant. -10 1.52 2.17 -1.74 -10VAS 0.1 9.6 6.4 4.1 0.9Eec. Torque (N.m) 84 69 83 121 136CPK (lUlL) 91 791 424 785 553OHP (umol/L) 56 130 57 45 297Cr(umollL) 5996 10315 7232 6769 9369OHPICr 9.3 12.6 7.9 6.7 31.7Exercise Duration Longer (300 reps)Test Session (Hour) pre-test 24 48 96 168DDS Soreness -10 13.29 10.74 14.24 -4.21DDS Unpleasant. -10 2.99 10.88 -1.70 -10VAS 0.0 8.4 9.8 5.0 2.8Eec. Torque (N.m) 128 78 70 102 104CPK (lUlL) 125 2043 4545 18070 2839Ol-IP (umollL) * 34 98 99 69Cr(umol/L) * 5170 8969 7581 7785OHP/Cr * 6.6 10.9 13.1 8.9192Subject 13Age 43Gender MWeight (Kg) 66Leg Tested RPhysical Activity (Hrlwk) 0Exercise Duration Shorter (180 reps)Test Session (Hour) pre-test 24 48 96 168DDS Soreness * 12.3 9.97 13.68 6.96DDS Unpleasant. -9.23 8.06 4.33 2.20 -3.49VAS 0.9 8.4 * 59 1.1Ecc. Torque(N.m) 153 57 83 77 115CPK (lU/L) 72 650 490 926 928OI-IP(umol/L) 68 84 80 81 100Cr (umol/L) 5932 8307 4932 6243 12590OI-IPICr 11.5 10.1 16.2 12.9 7.9Exercise Duration Longer (300 reps)Test Session (Hour) pre-test 24 48 96 168DDS Soreness -9.52 13.29 10.00 12.03 -9.47DDS Unpleasant. -10 1.83 1.58 1.00 -10VAS 0.2 8.4 5.9 2.5 0.2Eec. Torque (N.m) 134 73 99 99 155CPK (lU/L) 38 354 224 181 140OI-IP(umol/L) 199 149 88 167 111Cr(umol/L) 8137 10799 5138 8384 5769OI-IP/Cr 24.5 13.8 17.1 19.9 19.2193Subject 14Age 52Gender FWeight (Kg) 58.1Leg Tested LPhysical Activity (Hr/wk) 2Exercise Duration Shorter (180 reps)Test Session (Hour) pre-test 24 48 96 168DDS Soreness -10 3.91 4.49 -8.30 -10DDS Unpleasant. -10 -8.50 -4.55 -10 -10VAS 0.0 1.8 3.6 0.2 0.0Ecc.Torque(N.m) 132 78 94 103 108CPK (lU/L) 42 82 53 32 163OHP(umol/L) 162 179 91 128 57Cr (umol/L) 8925 8832 8223 6639 5887OHP/Cr 18.2 20.3 11.1 19.3 9.7Exercise Duration Longer (300 reps)Test Session (Hour) pre-test 24 48 96 168DDS Soreness -10 1.62 3.82 -10 -10DDS Unpleasant. -10 -9.71 -2.15 -10 -10VAS 0.0 1.8 3.4 0.0 0.0Eec. Torque (N.m) 132 91 100 105 92CPK (lUlL) 27 195 136 68 38OHP(umolIL) 96 193 185 104 82Cr(umollL) 5001 11929 9331 9098 5813OHPICr 19.2 16.2 19.8 11.4 14.1194Subject 15Age 20Gender FWeight (Kg) 93.1Leg Tested LPhysical Activity (Hr/wk) 2Exercise Duration Shorter (180 reps)Test Session (Hour) pre-test 24 48 96 168DDS Soreness -10 11.89 11.92 13.27 -10DDS Unpleasant. -10 10.96 11.42 1.52 -10VAS 0.0 8.9 9.1 2.7 0.0Ecc. Torque (N.m) 125 69 62 115 162CPK (lUlL) 46 * 102 178 159Ol-IP (umol/L) 47 195 88 159 209Cr(umollL) 5088 10484 5790 9186 10892OHPICr 9.24 18.6 15.2 17.3 19.2Exercise Duration Longer (300 reps)Test Session (Hour) pre-test 24 48 96 168DDS Soreness -10 12.56 11.39 -0.55 -10DDS Unpleasant. -10 1.55 6.5 -8.85 -10VAS 0.0 6.4 6.4 0.9 0.0Eec. Torque (N.m) 101 111 116 160 165CPK (lUlL) 60 192 112 68 51OHP(umollL) 155 255 143 171 132Cr(umol/L) 9475 17781 11685 8131 8589OHPICr 16.4 14.3 12.2 21.0 15.4195Subject 16Age 34Gender MWeight (Kg) 74.9Leg Tested RPhysical Activity (Hr/wk) 2Exercise Duration Shorter (180 reps)Test Session (Hour) pre-test 24 48 96 168DDS Soreness -10 13.82 13.40 14.05 -10DDS Unpleasant. -10 1.26 11.68 3.70 -10VAS 0.0 4.2 5.9 2.3 0.0Ecc. Torque (N.m) 236 176 159 223 266CPK (lUlL) 137 380 188 79 86OHP (umol/L) 36 73 63 53 20Cr(umollL) 4688 12132 14462 4959 3134OHPICr 7.7 6.0 4.4 10.7 6.4Exercise Duration Longer (300 reps)Test Session (Hour) pre-test 24 48 96 168DDS Soreness -10 8.55 14.23 5.41 -10DDS Unpleasant. -10 0.53 8.15 -8.56 -10VAS 0.0 3.6 7.1 2.1 0.0Ecc. Torque (N.m) 209 167 200 256 231CPK (lUlL) 106 755 356 104 81Ol-IP (umollL) 82 72 74 79 66Cr(umol/L) 10128 7442 6722 9926 7041OI-IPICr 8.1 9.7 11.0 7.9 9.4196Subject 17Age 23Gender FWeight (Kg) 59Leg Tested RPhysical Activity (Hr/wk) 3Exercise Duration Shorter (180 reps)Test Session (Hour) pre-test 24 48 96 168DDS Soreness -10 9.33 12.12 4.27 -4.24DDS Unpleasant. -10 -0.55 3.18 -3.70 -7.86VAS 0.0 4.1 5.0 3.2 0.0Ecc.Torque(N.m) 128 72 74 87 100CPK (lUlL) 20 120 80 90 120OI-IP (umol/L) 60 198 62 167 162Cr (umollL) 3536 7319 3855 8129 9246OHPICr 16.9 27.0 16.1 20.5 17.5Exercise Duration Longer (300 reps)Test Session (Hour) pre-test 24 48 96 168DDS Soreness -10 10.08 11.62 9.26 -7.52DDS Unpleasant. -10 0.86 3.08 0.05 -10VAS 0.0 3.7 5.5 1.8 0.0Eec. Torque (N.m) 128 87 85 88 108CPK (lUlL) 20 162 104 131 61Ol-IP (umol/L) 185 96 170 * 168Cr (umollL) 7561 6699 9462 7454 7261OHPICr 24.5 14.3 17.9 * 23.1197Subject 18Age 40Gender FWeight (Kg) 50Leg Tested RPhysical Activity (Hr/wk) 2Exercise Duration Shorter (180 reps)Test Session (Hour) pre-test 24 48 96 168DDS Soreness -10 12.26 7.39 -4.67 -10DDS Unpleasant. -10 2.76 -1.26 -7.50 -10VAS 0.0 5.0 3.2 0.7 0.0Ecc. Torque (N.m) 68 37 45 60 63CPK (lU/L) 20 34 20 20 20OHP (umol/L) 68 21 38 54 62Cr(umol/L) 6836 4167 4170 6956 7830OI-IP/Cr 9.9 5.0 9.1 7.8 7.9Exercise Duration Longer (300 reps)Test Session (Hour) pre-test 24 48 96 168DDS Soreness -8.99 -0.30 -9.33 -7.30 -7.08DDS Unpleasant. -10 -6.88 0.00 -8.47 -8.33VAS 0.0 5.0 4.0 0.7 0.0Ecc. Torque (N.m) 35 41 47 49 49CPK (lU/L) 29 48 23 20 20OHP (umol/L) 128 69 60 50 77Cr(umol/L) 5313 5184 6335 4103 7976OI-IP/Cr 24.1 13.3 9.5 12.2 9.6198Subject 19Age 24Gender FWeight (Kg) 54.5Leg Tested RPhysical Activity (Hr/wk) 3Exercise Duration Shorter (180 reps)Test Session (Hour) pre-test 24 48 96 168DDS Soreness -10 8.12 7.92 4.70 -10DDS Unpleasant. -9.09 2.35 2.52 -2.06 -10VAS 0.1 5.0 5.3 1.8 0.1Ecc. Torque (N.m) 80 37 34 83 103CPK (lUlL) 43 165 82 60 68OHP(umolIL) 99 121 72 167 50Cr(umol/L) 4679 8416 5949 7351 2835OHPICr 21.2 14.4 12.1 22.7 17.6Exercise Duration Longer (300 reps)Test Session (Hour) pre-test 24 48 96 168DDS Soreness -10 8.12 7.92 4.70 -10DDS Unpleasant. -10 3.12 3.53 -6.64 -8.49VAS 0.0 7.3 8.4 1.2 0.0Eec. Torque (N.m) 82 54 66 81 78CPK (lUlL) 40 108 58 62 47Ol-IP (umol/L) * * 37 102 147Cr (umollL) * 5549 3038 * *OHPICr * * 12.2 * *IndividualSubjectData-ChapterTwo*=missingdataIndividualTc-99mWBCdataisincludedinAppendixFourSubject1Age33GenderFWeight (Kg)54.0LegTestedRPhysicalActivity(Hr/wk)4.5ExerciseDurationLonger(300 reps)TestSession(Hour)pretest2420244872DDSSoreness-7.970.307.6813.5014.9114.6210.29DDSUnpleasant.-10-6.61-5.15-0.73-1.71-0.67-0.40ROM(degrees)140140140135135130135VL(slope)-0.280-0.259-0.274-0.133-0.110-0.118-0.121VM(slope)-0.252-0.236-0.269-0.106-0.095-0.09-0.145TestSession(Hour)pretest02420244872Ecc.Torque(N.m)11759757958607285I— 0 0Subject2Age21GenderFWeight (Kg)70LegTestedRPhysicalActivity(Hr/wk)5ExerciseDurationLonger(300reps)TestSession(Hour)pretest2420244872DDSSoreness1.526.895.585.2611.5910.4611.23DDSUnpleasant.-5.27-3.80-3.493.76-1.36-1.68-0.82ROM(degrees)135135135135130130135VL(slope)-0.403-0.586-0.407-0.437-0.370-0.353-0.399VM(slope)-0.414-0.497-0.404-0.719-0.856-0.299-0.723TestSession(Hour)pretest02420244872Ecc.Torque(N.m)138101149141133107153136Subject3Age28GenderFWeight (Kg)72.7LegTestedRPhysicalActivity(Hr/wk)5ExerciseDurationLonger(300reps)TestSession(Hour)pretest2420244872DDSSoreness-1012.099.9714.9411.3316.115.20DDSUnpleasant.-10-4.32-3.426.410.962.96-0.99ROM(degrees)135135130125125125130VL(slope)-0.328-0.329-0.372-0.300-0.269-0.260-0.294VM(slope)-0.253-0.338-0.354-0.279-0.228-0.210-0.196TestSession(Hour)pretest02420244872Eec.Torque(N.m)99571089973707383N)Subject4Age23GenderFWeight(Kg)47.7LegTestedRPhysicalActivity(Hr/wk)1ExerciseDurationLonger(300reps)TestSession(Hour)pretest2420244872DDSSoreness-9.524.416.429.6511.736.03-6.79DDSUnpleasant.-10-8.97-7.21-5.79-1.86-8.27-10ROM(degrees)140135135140135140140VL(slope)-.337-.249-.238-.188-.112-.155-.213VM(slope)-.262-.232-.190-.142-.073-.068-.226TestSession(Hour)pretest02420244872Ecc.Torque(N.m)8865606153567177I\.)r’.)Subject5Age29GenderFWeight (Kg)52.3LegTestedRPhysicalActivity(Hr/wk)4.5ExerciseDurationLonger(300reps)TestSession(Hour)pretest2420244872DDSSoreness-8.7414.8612.1413.8014.5010.7911.88DDSUnpleasant.-10-1.50-1.331.002.17-3.08-2.79ROM(degrees)145140140140135145145VL(slope)-.100-.227-.232-.197-.195-.224-.210VM(slope)-.278-.230-.640-.291-.295-.449-.299TestSession(Hour)pretest02420244872Ecc.Torque(N.m)8659768665657484cJ3Subject6Age26GenderFWeight(Kg)60.5LegTestedRPhysicalActivity(Hr/wk)4ExerciseDurationLonger(300 reps)TestSession(Hour)pretest2420244872DDSSoreness-9.47-7.61-8.79-2.6410.3612.33-9.18DDSUnpleasant.-10-6.79-6.264.7710.6412.82-9.80ROM(degrees)140140140135130130140VL(slope)-.106-.110-.058-.026-.050-.043-.085VM(slope)-.139-.144-.024.028.002-.035-.067TestSession(Hour)pretest02420244872Ecc.Torque(N.m)4229415648386754Subject7Age20GenderFWeight (Kg)64.0LegTestedRPhysicalActivity(Hr/wk)1.5ExerciseDurationLonger(300reps)TestSession(Hour)pretest2420244872DDSSoreness-9.1410.6510.387.5210.305.52-1.29DDSUnpleasant.-10-0.420.921.963.300.47-7.52ROM(degrees)140130135135135135140VL(slope)-0.140-0.218-0.234-0.189-0.169-0.231-0.226VM(slope)-0.139-0.232-0.194-0.150-0.135-0.195-0.184TestSession(Hour)pretest02420244872Eec.Torque(N.m)13263116118121126171167I\.)cD ci,Subject8Age30GenderFWeight (Kg)65.0LegTestedRPhysicalActivity(Hr/wk)3ExerciseDurationLonger(300reps)TestSession(Hour)pretest2420244872DDSSoreness-0.911.502.303.293.470.912.56DDSUnpleasant.-4.05-2.920.921.963.300.47-7.52ROM(degrees)135135135135135135135VL(slope)-0.143-0.159-0.118-0.097-0.044-0.093-0.109VM(slope)-0.024-0.1310.059-0.0450.0320.020-0.041TestSession(Hour)pretest02420244872Eec.Torque(N.m)1331048010981108121115N)Subject9Age29GenderFWeight (Kg)62.0LegTestedRPhysicalActivity(Hr/wk)1.5ExerciseDurationLonger(300reps)TestSession(Hour)pretest2420244872DDSSoreness-9.6713.3514.3214.6215.0814.8815.73DDSUnpleasant.-10-3.470.53-0.262.144.623.17ROM(degrees)150150150140145140145VL(slope)-0.314-0.384-0.420-0.334-0.421-0.235-0.368VM(slope)-0.277-0.076-0.065-0.198-0.193-0.208-0.260TestSession(Hour)pretest02420244872Eec.Torque(N.m)11269978964537584r’)Subject10Age27GenderFWeight (Kg)54.5LegTestedRPhysicalActivity(Hr/wk)3ExerciseDurationLonger(300reps)TestSession(Hour)pretest2420244872DDSSoreness5.177.928.889.477.8610.949.77DDSUnpleasant.-8.392.474.445.947.206.247.02ROM(degrees)135128130125125128133VL(slope)-0.345-0.413-0.430-0.319-0.314-0.324-0.247VM(slope)-0.143-0.183-0.188-0.255-0.172-0.278-0.266TestSession(Hour)pretest02420244872Ecc.Torque(N.m)12253585661566057C

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