Muscle fascicle behavior during eccentric cycling and its relation to muscle soreness.

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Muscle Fascicle Behavior during Eccentric Cycling and Its Relation to Muscle Soreness ˜ AILILLO1,2, ANTHONY J. BLAZEVICH1, and KAZUNORI NOSAKA1 LUIS PEN 1

School of Exercise and Health Sciences, Centre for Exercise and Sports Science Research, Edith Cowan University, Joondalup, WA, AUSTRALIA; and 2Exercise Science Laboratory, School of Kinesiology, Faculty of Medicine, Universidad Finis Terrae, Santiago, CHILE

ABSTRACT ˜ AILILLO, L., A. J. BLAZEVICH, and K. NOSAKA. Muscle Fascicle Behavior during Eccentric Cycling and Its Relation to Muscle PEN Soreness. Med. Sci. Sports Exerc., Vol. 47, No. 4, pp. 708–717, 2015. A single bout of eccentric exercise confers a protective effect against muscle damage and soreness in subsequent eccentric exercise bouts, but the mechanisms underpinning this effect are unclear. Purpose: This study compared vastus lateralis (VL) muscle–tendon behavior between two eccentric cycling bouts to test the hypothesis that muscle–tendon behavior would be different between bouts and would be associated with the protective effect. Methods: Eleven untrained men (27.1 T 7.0 yr) performed two bouts of eccentric cycling (ECC1 and ECC2) separated by 2 wk for 10 min at 65% of maximal concentric workload (191.9 T 44.2 W) each. Muscle soreness (by visual analog scale) and maximal voluntary isometric contraction (MVC) torque of the knee extensors were assessed before and 1–2 d after exercise. Using ultrasonography, VL fascicle length and angle changes during cycling were assessed, and tendinous tissue (TT) length changes were estimated. VL EMG amplitude, crank torque, and knee joint angles were measured during cycling. Results: Soreness was greater (P G 0.0001) after ECC1 than ECC2, although MVC changes were not different between bouts (P = 0.47). No significant differences in peak EMG amplitude (normalized to EMG during MVC), crank peak torque, or knee angles were evident between bouts. However, fascicle elongation was 16% less during ECC2 than ECC1 (P G 0.01), indicating less fascicle strain in ECC2. Maximum TT length occurred at a smaller knee joint angle during ECC2 than ECC1 (P = 0.055). Conclusion: These results suggest that a lesser fascicle elongation and earlier TT elongation were associated with reduced muscle soreness after ECC2 than ECC1; thus, changes in muscle–tendon behavior may be an important mechanism underpinning the protective effect. Key Words: LENGTHENING CONTRACTIONS, FASCICLE LENGTH, MUSCLE–TENDON BEHAVIOR, DELAYED ONSET MUSCLE SORENESS, TENDINOUS TISSUE, REPEATED BOUT EFFECT

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‘‘repeated bout effect’’ (35). McHugh (29) suggested that neural, mechanical, and cellular adaptations may occur in response to the initial bout, and thus the mechanisms underpinning the repeated bout effect were likely multifactorial; however, the specific mechanisms have yet to be defined. It is well documented that eccentric exercise-induced muscle damage is triggered by muscle strain, resulting in connective tissue damage and the up-regulation of Ca2+-mediated myocellular processes leading to myofilament and membrane damage (3,11,15,21). Muscle strain during eccentric contractions is considered the primary cause of changes in indirect markers of muscle damage such as DOMS and the loss of muscle function (14,28). Nonetheless, it has been speculated that passive and/or dynamic muscle stiffness increases after the first eccentric exercise bout, which reduces muscle strain and confers a protective effect against subsequent eccentric exercise-induced muscle damage in subsequent bouts (14,29,30). If reductions in muscle strain do occur after the first eccentric exercise bout, it is possible that muscle fibers lengthen less and

xercise incorporating eccentric (lengthening) contractions induces muscle damage that is characterized by delayed onset muscle soreness (DOMS) and prolonged loss of muscle function, especially when it is performed by muscles that have not been exposed to eccentric contractions regularly (10). However, when the same eccentric exercise is performed within several weeks, DOMS is minimal and the recovery of muscle function is faster (35,37). This adaptation conferred by a single bout of eccentric exercise is called the Address for correspondence: Luis Pen˜ailillo, School of Kinesiology, Universidad Finis Terrae, 1509 Pedro de Valdivia Av., Providencia, Santiago, Chile. E-mail: [email protected]. Submitted for publication March 2014. Accepted for publication August 2014. 0195-9131/15/4704-0708/0 MEDICINE & SCIENCE IN SPORTS & EXERCISEÒ Copyright Ó 2014 by the American College of Sports Medicine DOI: 10.1249/MSS.0000000000000473

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Copyright © 2015 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.

METHODS Participants. Eleven men who had not performed lower limb resistance training regularly in the past 6 months and who reported no history of neurological disorders or orthopedic lower limb injuries completed an informed written consent form and a medical questionnaire before participating in the study. The sample size was calculated on the basis of an > level of 0.05 and a power (1 j A) of 0.8, with an estimated 20% difference in fascicle length change between the first and second eccentric cycling bouts using the VL fascicle length data of a previous study (12). Ethical approval was obtained from the institutional Human Research Ethics Committee before study commencement. The participants’ age, height, body mass, body mass index, and peak oxygen consumption during concentric cycling exercise (mean T SD, n = 11) were 27.1 T 7.0 yr, 178.4 T 8.2 cm, 73.6 T 6.2 kg, 23.1 T 1.3 kgImj2, and 3.3 T 0.6 LIminj1, respectively. Participants were

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instructed not to perform any exercise, take anti-inflammatory medication, or apply any treatments (e.g., massage, stretching) from 2 d before to 2 d after each cycling bout. Study design. The participants reported to the laboratory for two 3-d blocks (one exercise day followed by two recovery days) separated by 2 wk because our previous study (37) showed a strong repeated bout effect of eccentric cycling for this period. The participants performed 10 min of eccentric cycling at the same intensity for each block (ECC1 and ECC2), and muscle soreness by visual analog scale (VAS) and maximal voluntary isometric contraction (MVC) torque of the knee extensors were assessed before and 1–2 d after each cycling bout. During cycling, VL fascicle behavior and the surface EMG, crank torque, and knee joint angle were recorded. Changes in muscle soreness and MVC torque and the variables measured during cycling were compared between bouts. Cycling exercise. An incremental cycling test to deter˙ O2peak was performed at least 1 wk before ECC1 mine V using a recumbent ergometer (Tunturi F30R, Almere, the Netherlands). The test started at a power output of 50 W for 4 min and increased 25 W every 1 min until volitional exhaustion. Cadence was kept at 60 rpm, and the participants received verbal encouragement during the test. Eccentric cycling was performed using a recumbent cycle ergometer fitted with a motor that moved the cranks of the ergometer backwards at a selected cadence (Metitur, Jyva¨skyla¨, Finland). Eccentric contractions were mainly performed by knee extensor muscles when the participants tried to resist the backward movement of the cranks. A 5-min familiarization period was performed at a low power output (50 W) before 10 min of cycling (60 rpm) set at 65% of the maximal power ˙ O2peak test. We have output (POmax) obtained during the V previously shown that 30 min of eccentric cycling at 60% POmax induced moderate muscle damage (37). On the basis of this and a pilot study, we assumed that 10 min of eccentric cycling at 65% POmax would be sufficient to induce significant soreness, especially after the first exercise bout. This exercise duration was also considered more likely to be performed in a clinical exercise setting in an initial exercise bout than the longer (e.g., 30 min) bouts used in previous studies (25,37). Crank torque and knee range of motion. Crank torque was continuously measured by a KIP power meter bicycle crank (Breakaway Innovations, Perth, Australia) implemented to the left crank at a sampling frequency of 477 Hz, and the data were stored in a data logger; the average power output was instantaneously calculated and displayed on a screen (Rider 50; Bryton, Taipei, Taiwan). The cycle ergometer was equipped with a switch (with a crank angle of 270-) that sent a pulse to the ultrasound apparatus and the PowerLab system (AdInstruments, Bella Vista, Australia), which allowed us to synchronize the ultrasound, EMG, crank torque, and knee joint angle data. After synchronization, ultrasound video clips, EMG, and crank torque data of three revolutions at 1 and 10 min of cycling were analyzed because muscle fascicle length changes were not significantly different between 10 revolutions, as shown below. Knee joint range of motion was continuously measured

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thus less muscle damage and soreness are elicited. However, to the best of our knowledge, no previous study has compared the muscle lengthening behavior between two successive bouts of eccentric exercise to investigate the repeated bout effect. The B-mode ultrasound technique has been used to examine human skeletal muscle behavior in vivo (16). In particular, several studies have used this technique to examine the behavior of muscle fascicles (fascicle length and angle) and tendinous tissue (TT; outer tendon + aponeurosis) of a muscle– tendon unit (proximal tendon + muscle + distal tendon (MTU)) during movements (6,16,22,23). Some studies have shown that MTU lengthening results mainly from TT elongation with a quasiisometric behavior of muscle fascicles during eccentric contractions in tibialis anterior (40) and gastrocnemius (17). Other studies have reported that vastus lateralis (VL) fascicles and TT were simultaneously lengthened during eccentric knee extensor contractions (12,23). These studies show that the ultrasound technique could detect a difference in muscle and tendon behavior during various tasks and therefore could be used to assess differences in muscle–tendon behavior between bouts of eccentric exercise. We have previously reported that an initial 30-min bout of eccentric cycling induced greater increases in muscle soreness and decreases in muscle function 1–3 d after eccentric cycling when compared with the second eccentric cycling bout performed 2 wk later (37). In eccentric cycling, many submaximal eccentric contractions (e.g., 60 rpm 10 min = 600 eccentric contractions) are performed, so it appears to be an ideal exercise model to investigate muscle damage occurring in sporting/exercise activities. Therefore, the present study examined VL muscle fascicle and tendon behavior during two eccentric cycling bouts performed 2 wk apart to test the hypotheses that muscle fascicle elongation would be less during the second eccentric cycling bout when compared with the initial bout, and that a smaller fascicle elongation during the second eccentric cycling bout would be associated with the repeated bout effect.

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by an electronic goniometer (SG, Biometrics Ltd., Ladysmith, VA). The data collected at different frequencies were resampled at 200 Hz to relate the changes in variables over a revolution and to estimate TT length changes in relation to knee joint angle changes. Before cycling for both ECC1 and ECC2, MVC torque was measured at 90- of knee flexion (0- = full extension) with 100- hip flexion on the ergometer using the KIP power meter crank to determine the relative torque level and VL EMG activity exerted during eccentric cycling in relation to VL EMG during the maximal knee extension torque measurement. Knee extension torque with EMG was measured three times with a 60-s rest between attempts, and the attempt with the highest torque value was used for further analyses. Electromyography. Neural adaptations have been speculated to partially explain the repeated bout effect (29). A previous study showed that EMG amplitude was lower during the second bout of eccentric cycling than the initial bout (37). Thus, surface EMG was recorded from VL during cycling. The skin was shaved and cleaned with alcohol swaps and electrodes were placed at 2/3 of the distance from the anterior superior iliac spine to the patella, according to SENIAM guidelines as shown in Figure 1. A Bagnoli-8 desktop EMG system (Delsys Inc., Natick, MA) was used to record EMG data using a bipolar electrode configuration (DE-2.1 SEMG sensor; Delsys Inc., Natick, MA) with a 10-mm interelectrode distance. Sampling frequency was set at 2000 Hz and a 10- to 450Hz digital band-pass filter was applied. EMG data were smoothed using a root mean square algorithm with a 50-ms window to produce a linear envelope from which the average peak amplitude and mean amplitude of the pushing and recovery phases of 10 consecutive revolutions were used for comparisons between bouts according to a previous study (30). Values are presented as relative (percentage) to the EMG peak amplitude obtained during an MVC at 90- of knee flexion assessed on the cycle ergometer before cycling. Muscle and tendon behavior. As shown in Figure 1, with each subject seated on the ergometer, an ultrasound probe

(60 mm, Model UST-5712, 7.5 MHz, B-mode; Aloka SSD >-10, Tokyo, Japan) was attached to the left mid-thigh just above the VL EMG electrode (explained above). The probe was firmly placed on the skin by surgical tape, and a custommade flexible plastic cast (size: 10 20 cm; surrounding the leg and secured by Velcro straps) was also used to minimize the movement of the probe. Echo absorptive tape was used to ensure that no probe displacement occurred during the 10-min cycling bouts. The ultrasound video clips were captured at a sampling rate of 47 Hz (i.e., 47 frames per pedal revolution) and analyzed offline with video analysis software (Tracker 4.7, free download: http://www.cabrillo.edu/~dbrown/tracker/). The superior and inferior aponeuroses and a clearly identifiable fascicle (i.e., visible echo from the fascicle interspaces that could be followed across the image) were identified to track the same fascicle throughout the movements and digitized frame by frame assuming a linear continuation of the fascicles (Fig. 2). For each subject, the entire length of the VL fascicle (Lf) was estimated using a trigonometric method as shown in Figure 2 (12,13) because Lf could not be visualized throughout the pedaling cycle. The error in the estimation of Lf using this method has been reported to be 2%–7% (12,13). To estimate the reliability of the fascicle length measures, 10 consecutive revolutions were analyzed for one subject. The average change in fascicle length (i.e., max–min) was calculated using averages of 2 to 10 revolutions. The interclass correlation coefficient was 0.995 (95% confidence interval, 0.994–0.996; P G 0.0001). When comparing the average of 2, 3, 4, I, 10 revolutions for changes in fascicle length, the mean T SD values were 4.8 T 0.3, 4.9 T 0.5, 4.9 T 0.4, 4.9 T 0.4, 4.8 T 0.4, 4.7 T 0.4, 4.8 T 0.5, 4.9 T 0.5, and 5.0 T 0.5 cm, respectively. Because no significant difference in the change in the fascicle length was evident, and considering the time constraints of manual frame-by-frame fascicle length analysis, the average of three consecutive revolutions was used to represent the average length change of 60 rpm. No significant difference in the fascicle length changes was observed over time (1–10 min); thus, the

FIGURE 1—Laboratory setup during data collection.

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results at 1 and 10 min of cycling are reported in the results. The fascicle length and angle data were filtered using a smoothing spline method (43). The resting fascicle length was also estimated using the ultrasound images taken when each subject was seated in the ergometer with the knee joint at 90- of flexion. Resting fascicle length was measured three times from three different images per subject, and the average of the three measures was used for further analyses. The length of TT (LTT) was defined as the sum of the proximal and distal tendinous structures and the aponeurosis (24,33). The length change in the TT (tendon and aponeurosis; LTT) was calculated as follows: LTT ¼ LMTU j Lf total cos 5

where LMTU is the MTU length and 5 is the angle between fascicle and deeper aponeurosis (17,24). LMTU changes were estimated using previously derived models on the basis of the knee joint position (angle) data and the limb length of each subject using the Hawkins and Hull model (20). MVC torque. The participants performed a 5-min warm-up on a cycle ergometer (Monark 828E, Vansbro, Sweden) with a load of 1 kPa at 60 rpm before testing. The MVC torque of the left knee extensors was measured at 70- of knee flexion (1) on an isokinetic dynamometer (Biodex, System 3, Shirley, NY). Once seated in the chair, the participants performed three submaximal contractions at 50%, 70%, and 80% of perceived MVC for 3 s each with a 1-min rest between contractions. Participants then performed three MVC with a 1-min rest between contractions, and the maximum value was used for further analyses. Before each eccentric cycling bout, three consecutive maximal concentric isokinetic contractions at 240-Isj1 were also performed to assess the knee joint angle at the peak concentric torque. The average of the three contractions was used for analyses.


Muscle soreness. Thigh muscle soreness was quantified using a 100-mm VAS in which 0 indicates no pain and 100 represents the worst pain imaginable (35). The participants were asked to mark the level of perceived pain of the quadriceps femoris muscles on the VAS while sitting down on and standing up from a 42-cm chair three times (2). Statistical analysis. A paired t-test was used to compare the average power output performed during each cycling bout. Because no significant difference in fascicle length changes was shown between 1 and 10 min of cycling by a two-way repeated-measures ANOVA, values from 1 and 10 min were averaged to represent the whole exercise to compare between ECC1 and ECC2. After synchronization of crank torque, EMG, muscle fascicle length, and TT length data, the average pattern of changes (mean T SD) of the pedal revolution are obtained. Paired t-tests were used to compare the changes in fascicle and TT length, average fascicle angle, peak cycling torque, peak EMG amplitude relative to the EMG during MVC measure, peak isokinetic concentric torque, and optimum angle between ECC1 and ECC2. A two-way repeated-measures ANOVA was used to compare changes in MVC torque and VAS for muscle soreness over time (before exercise and 1 and 2 d postexercise) between ECC1 and ECC2. If a significant main effect was found, a post hoc Fisher’s least significant difference test was performed. A significance level was set at P G 0.05. All statistical analyses were performed with PASW Statistics 21 software for Mac (SPSS Inc., IBM Company, Chicago, IL). Data are presented as mean T SD.

RESULTS MVC torque and muscle soreness. MVC knee extensor torque at baseline was not different between bouts (ECC1 = 270.3 T 49.9 and ECC2 = 275.3 T 47.9 NIm, P = 0.24). Figure 3A shows that muscle soreness was greater after



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FIGURE 2—Estimation of VL fascicle length and fascicle behavior during eccentric cycling at 20-, 80-, and 120- of knee flexion.

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FIGURE 3—Normalized changes in MVC torque from baseline (A) and changes in muscle soreness assessed by a VAS (B) before (pre) and 1–2 d after the first (ECC1) and second (ECC2) eccentric cycling bouts. *Significant (P G 0.05) interaction effect. #Significantly (P G 0.05) different from ECC1.

ECC2 than ECC1 at 1 d (P = 0.001) and 2 d (P = 0.003) postexercise. As shown in Figure 3B, no significant difference was evident for the changes in MVC torque between ECC1 and ECC2 (P = 0.47). The maximal voluntary isokinetic concentric peak torque before eccentric cycling (ECC1, 114.5 T 21.5 NIm; ECC2, 115.1 T 20.5 NIm; P = 0.85) and the optimum angle (ECC1, 61.9- T 10.3-; ECC2, 63.7- T 10.1-; P = 0.53) were similar between ECC1 and ECC2. Knee joint range of motion. The range of motion of the knee joint during cycling was not different between cycling bouts (P = 0.27), and it ranged from 18.8- T 8.7- to 116.8- T 4.4- (0- full extension). Cycling power output and peak crank torque. Average cycling power output was not different (P = 0.19) between ECC1 (189.7 T 43.2 W) and ECC2 (194.0 T 48.5 W). Crank torque increased during the pushing phase of the revolution and reached the peak at approximately 70- knee flexion for ECC1 and ECC2 and decreased in the recovery phase (Fig. 4A). Peak torque generated during cycling was not different (P = 0.17) between ECC1 (73.5 T 17.3 NIm) and ECC2 (79.5 T 19.1 NIm) as shown in Figure 4B. The peak torque generated during cycling was 29.5% T 11.3% and 33.1% T 14.2% of the single-leg MVC (248.7 T 44.0 and 240.4 T 45.4 NIm) for ECC1 and ECC2, respectively. EMG. The EMG amplitude during the MVC before exercise was not different between ECC1 and ECC2 (0.25 T 0.23 and 0.19 T 0.07 mV, respectively). Muscle activity increased during the revolution with increasing torque, and EMG amplitude peaked at ~70- knee flexion for ECC1 and ECC2, followed by gradual decreases during the recovery phase (Fig. 4C). As shown in Figure 4D, EMG peak amplitude was not significantly different (P = 0.13) between ECC1 (25.6% T 15.5% of MVC amplitude) and ECC2 (24.4% T 7.9% of MVC amplitude). When pushing and recovery phases (first and second half periods of the revolution, respectively) were analyzed separately, the EMG amplitude during pushing phase was not different between ECC1 and ECC2 (P = 0.09), but the EMG amplitude during the recovery phase was 60% smaller (P = 0.015) during ECC2 than ECC1 (Fig. 5). Fascicle and TT behavior. The resting fascicle length at 90- of knee flexion was not different (P = 0.28) between ECC1 (14.1 T 3.0 cm) and ECC2 (14.2 T 3.8 cm). The CV of the six revolutions used for analyses was 5.9% T 1.8%

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(range, 2.6%–8.1%), which was similar to that reported in a previous study (16). As shown in Figure 4E, the fascicle length increased as the knee joint angle increased, resulting in maximum fascicle lengths at 115.7- T 4.6- and 111.8- T 5.9of knee flexion during ECC1 and ECC2, respectively (without significant difference between bouts) and returning to the resting length when the knee moved into knee extension. No significant difference between ECC1 and ECC2 was evident for the knee angle at which maximum fascicle length occurred (P = 0.09). Fascicle length changed from 8.8 T 2.7 to 16.8 T 4.8 cm during ECC1 and from 7.7 T 2.5 to 14.4 T 5.0 cm during ECC2, although the knee moved similarly from ~20to 120- flexion in both bouts. The minimum (at ~20- of knee flexion) and maximum (at ~120- of knee flexion) fascicle lengths were significantly greater during ECC1 than ECC2 (P = 0.007 and P = 0.0008, respectively). Also, the magnitude of the fascicle length change (i.e., the difference between the minimum and maximal fascicle lengths) was greater (P = 0.017) during ECC1 (8.0 T 2.6 cm) compared with ECC2 (6.7 T 3.2 cm), as shown in Figure 4F. The maximum fascicle length was observed at low torque (ECC1, j0.14 T 8.6 NIm; ECC2, j2.04 T 9.3 NIm) and muscle activity (ECC1, 8.3% T 6.5% of MVC; ECC2, 3.0% T 3.0% of MVC) levels for both bouts. Peak torque was achieved at a similar knee joint angle (ECC1, 71.5- T 10.7-; ECC2, 72.6- T 10.2-; P = 0.5) and at a similar fascicle length (ECC1, 14.7 T 3.4 cm; ECC2, 13.5 T 3.1 cm; P = 0.09) between bouts. Fascicle angle changes were similar (P = 0.37) between bouts and increased significantly from 8.4- T 2.7- to 14.2- T 4.6- during ECC1 (P G 0.0001) and from 7.8- T 3.1- to 13.3- T 4.6- during ECC2 (P G 0.0001). The TT length increased as the knee joint moved toward maximum flexion, as shown in Figure 4G. There was a tendency (P = 0.055) for the maximum TT length to change at a greater knee angle during ECC1 (114.7- T 4.3-) when compared with ECC2 (111.3- T 6.9-). However, the maximum changes in TT length were not significantly different between bouts (P = 0.36) as shown in Figure 4H. Individual crank torque changes and fascicle and TT length changes were averaged for the 11 participants during ECC1 and ECC2 separately, and the averaged changes in fascicle (Fig. 6A) and TT (Fig. 6B) lengths are plotted against crank torque for ECC1 (solid line) and ECC2 (point line). The



BASIC SCIENCES FIGURE 4—Changes (mean T SD) in crank torque (A), VL EMG amplitude relative to the EMG amplitude during maximal isometric contractions at 90- of knee flexion (C), VL fascicle length (E), and TT length (G) in relation to knee joint angle during one pedal revolution, and peak crank torque (B), peak EMG amplitude relative to the EMG amplitude during MVC (D), maximal change in fascicle (F), and TT (H) lengths during the first (ECC1) and second (ECC2) eccentric cycling bouts. The SD of ECC1 and ECC2 are shown in dark and light gray shadows, respectively, in panels A, C, E, and G. *Significantly (P G 0.05) different from ECC1.

ascending segment of the curve shows that the fascicle elongation was not different (P = 0.09) at peak torque production between ECC1 and ECC2, but the maximal fascicle length was observed at the descending segment of the curve when little torque was produced. The fascicle length was smaller during ECC2 than ECC1 when both the knee joint was close to maximal flexion, and almost no crank torque was generated, which is shown by the dashed area (Fig. 6A). The TT length where the knee joint was close to maximal flexion and negative crank torque was developed was greater during ECC2


than ECC1 (Fig. 6B). Peak crank torque was achieved at smaller TT length during ECC1 than ECC2, and after that, the TT length tended to be greater during ECC2 than ECC1 as shown in the dashed area.

DISCUSSION The present study compared in vivo VL muscle fascicle and TT behavior during two eccentric cycling bouts (ECC1, ECC2) performed 2 wk apart to test the hypothesis that the



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FIGURE 5—Average VL EMG amplitude (mean T SD) relative to the EMG amplitude during MVC during the pushing (A) and recovery (B) phases for the first (ECC1) and the second (ECC2) eccentric cycling bouts. *Significantly (P G 0.05) different from ECC1.

magnitude of fascicle elongation during and muscle damage after ECC2 would be less than in ECC1. The two eccentric cycling bouts were performed without significant differences in the average power output, peak crank torque, EMG peak amplitude, or knee range of motion; however, the magnitude of fascicle elongation was 16% smaller during ECC2 than during ECC1 (Fig. 4E and F) and muscle soreness was developed only after ECC1 (Fig. 3B). These results support the hypothesis that fascicle elongation would be smaller in a repeated bout of eccentric cycling and associated with the protective effect. It is possible that the smaller muscle strain during ECC2 than ECC1 was a causative factor in the reduction in muscle soreness after ECC2. Our previous study (37), in which 30 min of eccentric cycling was performed at 60% of the concentric POmax by young healthy men, revealed an 11%–12% decrease in MVC torque 1–2 d after the first and second bouts, but the recovery of MVC torque was significantly faster after the second than the first bout. The nonsignificant changes in MVC torque in the present study (Fig. 3A) were likely due to the shorter exercise duration (10 min), and this was probably the reason why the repeated bout effect was not shown for MVC torque. The shorter bout was chosen for the ethical reason to minimize the burden to the participants and also because a shorter duration exercise is likely to be used in a clinical setting so we were able to determine whether changes in muscle–tendon behavior might underpin the effect in this specific circumstance. Indeed, we found that a typical repeated bout effect was observed for muscle soreness such that moderate DOMS was developed only after ECC1 (Fig. 3B). It has been

documented that DOMS is more associated with damage/ inflammation to connective tissue surrounding muscle fibers and fascia (36). It is possible that the 10-min eccentric cycling affected the connective tissue, but its effect on muscle fibers was minimal. As discussed below, it may be that the lesser fascicle length changes in ECC2 were associated with the lesser muscle soreness. Changes in crank torque during cycling were similar between bouts (Fig. 4A). The crank peak torque recorded during both ECC1 and ECC2 was ~30% of the MVC torque measured at 90- of knee flexion on the ergometer and also ~30% of the isometric MVC torque measured at 70- of knee flexion (Fig. 4B). The pattern of change in EMG amplitude (Fig. 4C) was similar to that of the crank torque, and peak VL EMG amplitude during eccentric cycling was also ~30% of MVC for both cycling bouts (Fig. 4D). These results indicate that eccentric contraction intensity during cycling was low and thus easily manageable in a clinical setting; however, it should be noted that more than 600 submaximal eccentric contractions were performed during cycling including the warm-up. Interestingly, EMG amplitude during the recovery phase of the cycle revolution was 60% smaller during ECC2 than ECC1 (Fig. 5B). This shows that VL was deactivated (i.e., it relaxed) faster during ECC2 than ECC1 in the recovery phase, which may be indicative of a more efficient motor pattern during ECC2 than ECC1. This could be associated with lesser muscle soreness because of the repeated bout effect. To the best of our knowledge, this was the first study to document muscle fascicle behavior during eccentric cycling. However, some studies have reported VL fascicle length

FIGURE 6—Relationship between fascicle (A) and TT (B) length changes and crank torque during a pedal revolution during the first (ECC1, solid line) and second (ECC2, dashed line) eccentric cycling bouts based on the average of all participants. Arrows represent the direction of the movement.

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be greater during ECC2 than ECC1 for the same total knee range of motion (i.e., same MTU length changes). However, TT elongation was not statistically different between bouts (Fig. 4G). This could be due to the assumptions associated with the calculation of MTU and TT lengths (20,24), which might not be sensitive enough to detect small differences between the conditions. It should be noted that the patella tracking could affect the VL mechanics, and this was not examined in the present study. However, it seems unlikely that patella movement differed substantially between bouts because the two bouts were separated by 2 wk without intervening exercises (training) or immobilization. It is noteworthy that TT continued to be stretched when fascicles were already at maximum length during both ECC1 and ECC2 (Figs. 4E and G) but that the maximal TT elongation was reached earlier during ECC2 (111.3- T 7.0-) compared with ECC1 (114.7- T 4.3-). These data suggest that TT tended to be elongated earlier and possibly to a greater extent (e.g., greater relative compliance) during ECC2 than ECC1. Thus, it is possible that TT elongated relatively more during ECC2 to ‘‘compensate’’ for the smaller fascicle elongation during ECC2 than ECC1. This could be investigated further when methodological advances allow for more accurate monitoring of tendon length changes. As shown in Figure 6, fascicle elongation did not occur during the torque generation phase (i.e., pushing phase) of the cycle revolution, and the maximal fascicle elongation occurred close to the maximal knee flexion position (i.e., long muscle length). Interestingly, this maximal fascicle elongation occurred when little torque was generated, as shown by the solid line in Figure 6A, but no further elongation occurred after the peak crank torque was achieved during ECC2 (dotted line). As mentioned above, the greatest fascicle elongation was observed at a low EMG activity for both cycling bouts, but EMG activity was significantly lower during the relaxation phase during ECC2 than ECC1 (Fig. 5B). This suggests that muscles were still activated when the fascicles were elongated during ECC1, but muscles were deactivated or relaxed for this phase of the revolution during ECC2, possibly representing an improved motor pattern. It is possible that greater fascicle elongation of activated muscles was responsible for the greater muscle soreness induced after ECC1 than ECC2. Although the present results do not provide a definitive explanation for the factors contributing to the 16% smaller fascicle elongation during ECC2 compared with ECC1 (Fig. 4F), some speculations can be made. It has been postulated that one adaptation to eccentric exercise is an increase in the number of sarcomeres in series, so that a given muscle length corresponds to a shorter sarcomere length, decreasing the overstretching of sarcomeres and muscle fiber damage (31,32). A shift in the optimum angle to a longer muscle length has been used as an evidence for sarcomerogenesis after an acute bout of eccentric exercise (7). However, a difference in the optimum angle assessed before ECC1 and ECC2 was not observed in the present study. If a shift of the



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changes during concentric cycling or isotonic/isokinetic eccentric contractions of the knee extensors. For example, Muraoka et al. (34) showed that fascicles operated at lengths between 9.1 T 0.7 and 12.7 T 0.5 cm during slow (40 rpm) concentric cycling in which the knee joint angle ranged from 39- to 114- of flexion. Guilhem et al. (19) reported that VL fascicle length changed from 9.3 to 12.8 cm during isotonic eccentric contractions (120% one-repetition maximum) and from 9.2 to 13.5 cm during maximal isokinetic (30-Isj1) eccentric contractions of the knee extensors from 30- to 90of knee flexion. Thus, the fascicle length changes during eccentric cycling found in the present study appear slightly greater than those reported during isotonic or isokinetic eccentric contractions in previous studies despite using a similar measurement technique (16,19), which could be due to the greater range of motion (120-) used presently than that in the previous studies (~90-). The present results showed that the fascicle angle changed from 8.4- T 2.7- to 14.2- T 4.6- during ECC1 and from 7.8- T 3.1- to 13.3- T 4.6during ECC2, similar to those reported by a previous study (42). Therefore, reasonably accurate and valid measurements of muscle fascicle length and angle appear to have been obtained in the present study. We found that fascicle length at ~20- of knee flexion was shorter during ECC2 (7.7 T 2.5 cm) than ECC1 (8.8 T 2.7 cm), but no significant difference between bouts was evident for the fascicle length measured at 90- of knee flexion before (i.e., resting) eccentric cycling. The fascicle length continued to increase until the knee joint was moved into maximal flexion (~120-) where the maximum fascicle length was observed. This maximum fascicle length was more than 2 cm greater during ECC1 (16.8 T 4.8 cm) than ECC2 (14.4 T 5.0 cm), as shown in Figure 4E. Importantly, the magnitude of fascicle length change (i.e., muscle strain) was significantly greater during ECC1 than ECC2 (Fig. 4F). It is speculated that the smaller fascicle elongation represents a lesser mechanical strain in muscle fascicles and muscle fibers, resulting in less DOMS. Nonetheless, it is not known whether this smaller muscle fascicle strain is reflective of a smaller sarcomere strain. Animal studies (8,9,18) have shown that for a given MTU length change (e.g., muscle strain of 5%), sarcomeres in different regions of a muscle work over different sarcomere lengths, experiencing different strains as they are stretched during eccentric contractions. One explanation for the eccentric contraction-induced muscle damage mechanisms is that some sarcomeres that extend beyond their working range are damaged (32). If this were the case, then one would hypothesize that a decrease in strain of some ‘‘weaker’’ sarcomeres is needed to produce a repeated bout effect. However, the present study examining adaptations at the fascicle level was not designed to test this hypothesis, so no conclusion can be made in this regard. Further studies are required to investigate the relationship between fascicle behavior and sarcomere mechanics. Because muscle fascicle elongation was smaller during ECC2 than ECC1, it was expected that TT elongation would

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optimum angle to a longer muscle length is an indicative of an increase in sarcomere number in series, then it does not appear that changes in muscle fiber length can explain the repeated bout effect observed in the present study. In support of this, as discussed previously, no difference in the resting fascicle length measured at 90- knee flexion before eccentric cycling was observed between ECC1 and ECC2. It may be that changes in the muscle’s dynamic stiffness could have contributed to the difference in the magnitude of fascicle elongation. Animal (41) and human (39) studies have shown that both passive and dynamic muscle stiffness were increased after a bout of downhill running and after 6 wk of isotonic eccentric training of the elbow flexors, respectively. McHugh (29) speculated that increased passive and dynamic muscle stiffness is one of the mechanisms underpinning the repeated bout effect. Increased muscle structural protein accretion 2–240 h after 30 stimulated eccentric contractions of the tibialis anterior has been reported in experimental animals (38). The authors hypothesized that reinforcement of the desmin cytoskeleton secondary to transcriptional up-regulation would provide mechanical protection from injury induced by eccentric contractions (38). Barash et al. (5) reported a threefold increase in muscle desmin protein content in the tibialis anterior of rats 7 d after a single bout of 30 eccentric contractions. In humans, increases in VL desmin protein content were found 14 d after 30 min of downhill running (11). Furthermore, increases in dystrophin, desmin, and titin mRNA levels in quadriceps femoris were found after the first downhill running bout in rats, but not after the second bout, and the authors speculated that sarcomere cytoskeletal remodeling was induced after the first bout (26). Lemos et al. (27) proposed, using mathematical and structural modeling of the changes in muscle–tendon length, that the aponeurosis is the major

contributor to reduced fascicle length changes when a muscle is elongated and speculated that one of the major functional roles of the aponeurosis was the protection of fascicles from excessive lengthening. It is also possible that tendon stiffness was decreased after the first bout, especially at the region near the muscle as has been reported in a previous animal study (4). Therefore, it may be that remodeling of muscle extracellular matrix was induced after ECC1, conferring a protective effect against ECC2 by increasing muscle stiffness. Further studies are however required to elucidate the molecular mechanisms of the muscle–tendon complex adaptations after a bout of eccentric exercise including eccentric cycling. In summary, the present study showed that VL muscle fascicles were elongated 16% less during the second than the first eccentric cycling bout performed 2 wk earlier. This change was associated with less muscle soreness after ECC2 than ECC1, which is consistent with the hypothesis that changes in muscle–tendon behavior may play a major role in the repeated bout effect. Further studies are necessary to determine why muscle fascicles lengthened less during ECC2 than ECC1 and how TT (including the aponeurosis) behaves in relation to the smaller fascicle elongation during secondary eccentric cycling bout. It is also necessary to investigate muscle–tendon behavior differences between bouts in other eccentric exercise modalities, such as isokinetic or isotonic eccentric contractions, which have been shown to induce a strong repeated bout effect.

The authors acknowledge Dr. Julien Louis and Dr. Eduardo Saez-Saez de Villareal for their help on the data collection and analyses. No funding was received. There were no conflicts of interests. The results of the present study do not constitute endorsement by the American College of Sports Medicine.

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Exercise-induced muscle soreness after concentric and eccentric isokinetic contractions.

Influence of preactivation on fascicle behavior during eccentric contraction.

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Leucine-enriched essential amino acids attenuate muscle soreness and improve muscle protein synthesis after eccentric contractions in rats.

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