Fascia Thickness Increases Following Eccentric Exercise Leading to Delayed Onset Muscle Soreness


 Background: The deep fascia fuses tightly with the skeletal muscle and, thus, may be damaged by eccentric loading. Methods: To study its possible involvement in delayed onset muscle soreness, 11 healthy participants (♂= 7; 24±2 years) performed fatiguing dumbbell elbow flexor eccentric exercise (EE) for one arm and concentric exercise (CE) for the other arm in random order and with random arm allocation. Before, immediately after and 24-96 hours post-exercise, maximal voluntary isometric contraction torque of the elbow flexors (dynamometer), pressure pain (algometer), palpation pain (100-mm visual analog scale), biceps brachii fascia thickness and fascia/muscle mobility during passive movement (both high-resolution ultrasound) were examined. Results: Palpation pain, suggestive of DOMS, was greater after EE than CE, and maximal voluntary isometric contraction torque decreased greater after EE than CE (p<.05). Relative to CE, EE increased fascia thickness at 48 (+17%), 72 (+14%) and 96 (+15%) hours post-exercise (p<.05). At 96 hours post-EE, the increase in fascia thickness correlated with palpation pain (r=.68; p<.05). Fascia mobility was not different between conditions, but compared to CE, muscle displacement increased at 24 (+31%), 72 (+31%), and 96 (+41%) hours post-EE (p<.05). Conclusion: Collectively, these results suggest an involvement of the deep fascia in delayed onset muscle soreness.

Hence, intramuscular force transmission is realized through the bers themselves and their endomysium.
A similar architecture-function relationship can be observed for the perimysium and the deep fascia.
Ultrastructural analyses have shown that the perimysium transmits lateral forces between muscle ber bundles due to direct tissue continuity [7]. Likewise, the deep fascia interacts with the underlying muscle via brous tissue expansions and direct ber insertions [8,9].
A recent systematic review with meta-analysis has shown that clinically diagnosed muscle injuries rarely affect the muscle tissue in an isolated manner [10]. In contrast, structural lesions mostly occur in the connective tissue, namely the myotendinous junction and the deep fascia. Considering that eccentric overload represents the main mechanism of injury, it has been hypothesized that the connective tissue, via its numerous links to muscular structures, absorbs and dampens excessive elongating forces [10].
Similar to muscle injury, delayed onset muscle soreness (DOMS) is predominantly induced by unaccustomed exercises with eccentric contractions. It could therefore be speculated that the associated tissue overstretch produces micro-lesions in the fascia which contribute to the development of DOMS at 24-72 hours post-exercise. This assumption is supported by both histological and experimental studies [11][12][13][14][15][16][17][18]. Due to its dense innervation and equipment with sensory receptors and free nerve endings that act as algogenic nociceptors [11,19], fascia appears to be of fundamental relevance in pain perception [13][14][15][16][17][18]. Schilder [17] investigated the pain sensitivity of different structures injecting hypertonic saline into the lumbar fascia and the erector spinae muscle, and showed that the irritation of the connective tissue evoked stronger pain responses than the muscle. Gibson [14] performed a similar experiment inducing DOMS by eccentric exercise in one leg. While injection of hypertonic saline into the exercised muscle did not increase pain sensitivity compared to the inactive control leg, a strong increase (exercised vs. control leg) was registered upon irritation of the fascia. Collectively, these ndings suggest that the fascia rather than muscle ber damage seems to cause DOMS. This is supported by data showing that structural muscle damage does not or only weakly correlate with the magnitude of DOMS [20][21][22].
Although fascia has a high nociceptive potential and may be vulnerable to mechanical overload (i.e., eccentric contractions), evidence on connective tissue involvement in DOMS is still scarce. The aim of the present study, therefore, was to investigate morphological changes in the biceps brachii fascia after eccentric exercise of the elbow exors. Since it is known that concentric contractions do not induce DOMS, morphological and functional changes in the fascia were compared between eccentric and concentric contractions. We hypothesized that, e.g. due to edema and swelling, fascia thickness would increase and fascia mobility would decrease after eccentric exercise only, and that the magnitude of the changes in the fascia would be associated with the magnitude of DOMS.

Study design and ethics
A randomized, controlled crossover trial with two exercise conditions was performed. At least 72 hours after a familiarization session, each participant visited the laboratory on 5 consecutive days. On day 1, eccentric and concentric exercise (randomized order and arm allocation) of the elbow exors was performed using a dumbbell. Outcome measures (see below) were assessed immediately before (base), immediately after (post 0) and every 24 hours up to 96-hours post-exercise (post 24, 48, 72, and 96). The study was approved by the Edith Cowan University Human Research Ethics Committee, and adhered to the Guidelines of Good Clinical Practice as well as the Declaration of Helsinki. All participants provided a written informed consent prior to the study participation.

Participants
Eleven (seven male and four female) healthy active students (24.3 ± 1.9 y; 167 ± 6 cm; 67.2 ± 8.1 kg; 293 ± 145 min sporting activity per week) volunteered to participate in the present study. The Edinburgh Handedness Inventory -Short Form [23] showed that all participants were right-handed. None of the participants had performed regular upper arm resistance training in the preceding six months. All individuals were healthy, not reporting any orthopedic, cardiovascular, neurological, endocrine or psychiatric diseases. Intake of anti-in ammatory or analgesic medicine was prohibited during or on the days before the experiment.

Determination of one-repetition maximum (1-RM)
Immediately before the exercise session (day 2), the one repetition maximum (1-RM) was measured using dumbbell curls (one arm for eccentric exercise, and the other arm for concentric exercise) according to the randomization (e.g., left arm eccentric, right arm concentric). This was done because a rst bout of eccentric exercise could protect against muscle damage in subsequent bouts of exercise [16,24]. After a warm-up with a light (50 % of the estimated 1-RM) dumbbell (10 elbow exion and extension movements), the participants performed either one isolated eccentric (lowering the dumbbell) or concentric (lifting the dumbbell) contraction with a load of approximately 80 % of the estimated 1-RM.
For the 1-RM assessment, each participant sat on a preacher arm-curl bench to stabilize their shoulder. For the eccentric 1-RM, the participant was instructed to lower the dumbbell from 90° elbow exion to full extension in three seconds, and for the concentric 1-RM, the participant lifted the dumbbell from full extension to 90° exion in three seconds. After each attempt, a one-minute rest was applied, and the procedure was repeated increasing the weight based on the perceived effort of the participant until a failed attempt occurred. All participants required less than ve attempts for each arm to determine the 1-RM.

Exercise protocols
The participants performed 6 sets of 10 repetitions of eccentric contractions for one arm and concentric contractions of the elbow exors for the other arm. Repetition duration was 3 seconds and the inter-set interval was 2 minutes. The exercises were performed with a dumbbell, using 80% of the individual 1-RM [25]. After each repetition, the investigator assisted in returning the dumbbell to its starting position to ensure eccentric-only or concentric-only contractions were performed with the load [26]. If necessary, the investigator carefully supported the participant to complete the protocol. After each set, the participants were asked to rate the effort of the exercise using the rating of perceived exertion (CR-10) scale ranging from 1 anchoring "extremely easy," to 10, "extremely hard" [27].

Outcome measures
The following measurements were taken from the exercised arm before (base), immediately after (excluding muscle pain measures) and 24, 48, 72 and 96 hours after exercise. To avoid possible in uences of circadian rhythm, examinations were performed at approximately the same time of the day for each participant. aligned with the dynamometer's axis of rotation ( Fig. 1). The elbow joint of the examined arm was positioned at 90° exion, and both shoulders were stabilized on the therapy bed. Following a brief warmup, each participant was instructed to ex the elbow joint as fast and strong as possible against the stationary lever arm for 3 seconds. Verbal encouragement was provided to elicit maximal effort for each attempt. Two attempts with a one-minute rest were performed. The average peak force, which could be maintained for 1 second, was considered as the MVIC torque and the mean of both measurements was used for further analysis [28, 29].

Pressure pain threshold (PPT)
PPT of the biceps brachii of the exercised arm was measured using an electronic algometer (Somedic AB, Sollentuna, Sweden). Each participant lay on a therapy bed in a relaxed supine position with the forearm being slightly elevated by a soft pad. The probe of the algometer (diameter of 1 cm) was placed on the biceps brachii muscle belly (9 cm above the elbow crease), and compressive force was gradually increased at a rate of approximately 50 kPa/s until the participant reported the rst sensation of pain.
The value (kPa) corresponding to the force applied at this moment was recorded. After a 10-second rest interval, the procedure was repeated twice, and the average of the two measures was used for further analysis.

Palpation pain
Muscle pain upon palpation of the elbow exors was assessed by a 100-mm visual analogue scale (VAS), anchoring 0 being no pain and 100 being worst possible pain [30,31]. Each participant was in the same position as that of PPT, and the measurement was taken from the same place as that of PPT (9 cm above the elbow crease). The investigator palpated the muscle of each participant with two ngers in longitudinal orientation [32]. The participants were asked to indicate the level of perceived pain by marking it on the VAS. Pressure was always applied by the same investigator and kept as constant as possible (approximately 400 kPa) between days and participants [28]. The measurement site was marked on the skin with a semi-permanent marker and renewed on a daily basis.

Fascia thickness
A high-resolution ultrasound (US) device (Aloka ProSound F75, Hitachi Healthcare, Tokyo, Japan) with an 8.0 x 1.5 cm linear array transducer was used (frequency range of 7.5 MHz, display depth of 3.0 cm, dynamic range of 60 dB and image gain of 50) to measure fascia thickness. Each participant was positioned in supine with the examined arm being attached to the lever arm of the isokinetic dynamometer at 35° exion (Fig. 1). The ultrasound probe was positioned longitudinally on the biceps muscle belly (with the center of the probe 9 cm above the elbow crease). The investigator ensured to stabilize the probe on the skin with minimal pressure in order to avoid compressing the tissue. Three static images were taken when the deep fascia was clearly recognizable as a hyperechoic region overlying the muscle. To ensure identical positioning during follow-up measurements, the US-transducer location was marked on the skin with a semi-permanent marker and renewed every day.
Fascia thickness was calculated using ImageJ (Image J 1.52 k software, USA). Within each US image, ve regions of interest (ROIs) at equidistant points were selected for the thickness measurement (Fig. 2a).
The average of the ve ROIs in each of the three images was chosen to determine the fascia thickness.

Fascia and muscle displacement
High-resolution ultrasound imaging was also used to evaluate fascia and muscle displacement during passive movements of the elbow joint of the exercised arm. Each participant was positioned as explained for the MVIC torque measure. The linear array ultrasound transducer was positioned longitudinally on the belly of the biceps brachii with the center of the probe at 9 cm above the elbow crease, and xed with elastic bandages and tape, ensuring the muscle was not compressed. The isokinetic dynamometer passively moved the elbow joint three times between 90° exion and full extension at an angular velocity of 5°/s (33). Previous studies have shown that re exive muscle contraction does not occur at this velocity [34,35]. Participants were instructed to remain completely relaxed, avoiding any voluntary muscle activity. In order to familiarize the participants with the device and measurement conditions, a warm-up of three exion-extension cycles was performed prior to the actual measurements [36,37]. Video recordings, depicting fascia and muscle tissue, were made via ultrasound at 10 Hz during the passive movement. Additionally, elbow joint position [°], relative to the neutral zero position, was recorded from the dynamometer signal to the laptop computer. Ultrasound videos were cut into extension and exion parts of the three repetitions using the synchronized joint position. If a participant could not reach full extension in the follow-up days due to increased muscle stiffness, all videos of this participant were cut to the lowest achieved extension angle.
The maximal horizontal displacement of biceps brachii fascia and muscle was quanti ed using a frameby-frame cross correlation algorithm proposed by Dilley [38]. To determine tissue displacement, rectangular ROIs were selected in the ultrasound videos. The used software [38] computes the correlation coe cient between the pixel gray levels of the consecutive frames. The pixel shift with the highest correlation coe cient represents the tissue displacement between the two successive frames. Three equidistant ROIs were de ned in the fascia starting 2.5 cm from the myotendinous junction. Additionally, three equidistant ROIs were de ned in the muscle on the same level. Horizontal tissue displacement was calculated by calculating the mean of three ROIs and three repetitions for the corresponding tissue ( Fig.  2b and 2c).

Statistical analyses
All data were tested for normal distribution using a Shapiro Wilk test and inference statistics were applied as appropriate. Baseline values were compared between conditions (eccentric vs. concentric) using a paired t-test or a Wilcoxon test. To examine the possible in uence of exercise order (eccentric or concentric arm rst), baseline values of participants starting with the respective conditions were compared by means of independent t tests or Mann-Whitney-U tests. As an additional check, we used point biserial correlation or contingency tables with Cramer-V to detect potential association of pre-post differences and treatment order.
Prior to the main analysis comparing between eccentric and concentric exercise for changes in the dependent variables over time, sphericity was checked by a Mauchly's test and the normal distribution of the residuals was examined by a Shapiro-Wilk test. If requirements were met, we performed 2-factorial ANOVAs (condition * time). Resulting effect sizes (η 2 ) were interpreted as small (0.01), medium (0.06) or large (0.14) according to Cohen (1988) [39]. In case of main effects for time, Bonferroni-Holm adjusted post hoc tests (paired t-test) were performed. Effect size interpretation followed the recommendations of Cohen (1988), distinguishing small (d = 0.2 to 0.5), medium (0.5 to 0.8) or large (0.8 or higher) effects [39]. In case of violations of the assumptions for parametric testing, Kubinger's bifactorial rank variance analyses with Bonferroni-Holm corrected post-hoc Wilcoxon tests were computed. We also investigated possible correlations between fascia thickness, fascia displacement, muscle displacement and palpation pain by a Pearson or Spearman correlation analysis. According to Cohen (1980), correlation coe cients (CC) were interpreted a small (r = 0.1 to 0.3), medium (0.3 to 0.5) or large (0.5 and higher) [40].
All analyzes were performed with SPSS 26 (SPSS Inc., Chicago, Illinois, USA) and BiAs Statistics (version 11.10, Goethe University, Frankfurt am Main, Germany). The level of signi cance was set to α = 0.05.

Results
All eleven participants completed the exercise protocol. However, the muscle/fascia displacement data of one participant had to be excluded due to an erroneous synchronization of the isokinetic testing system and the ultrasound device. No differences in the baseline values between the two exercise sessions were evident, and no intervention order effects were found (p > .05).

PPT
The 2x4 ANOVA for PPT did not reveal interactions or main effects for condition (p > 0.05), but a main

Palpation pain
The 2x4 bifactorial-rank-variance analysis for pain upon palpation revealed no interaction effect (p > 0.05). However, a signi cant main effect was found for time (p = 0.01) and condition (p < 0.001, Fig. 10). Collectively, these data suggest that EE consistently induced stronger pain (Fig. 4).

Fascia and muscle displacement
The 2x5 bifactorial-rank-variance analysis revealed no interaction effect for either fascia or muscle displacement (p > 0.05; Fig. 6, Table 1). However, regarding muscle displacement, a signi cant main

Correlations
Spearman's rank correlation showed no signi cant association of pre-post differences in VAS and fascia thickness for CE. Contrarily, increased fascia thickness following EE correlated strongly with palpation pain at 96-hours post-exercise (r = 0.682, p = 0.021). Additionally, at 72-hours post-exercise, there was a tendency for a correlation (p = 0.051) with a high positive correlation coe cient (r = 0.60).
Regarding tissue displacement measures, only muscle displacement correlated with VAS at post24 (r=-0.675; p = 0.032) and post96 (r=-0.685; p = .029) after CE. Yet, there was a tendency for an association of muscle displacement and VAS pain at post48 after EE (r = 0.626; p = 0.053).

Discussion
Skeletal muscle ber damage and in ammation have often been discussed as primary sources of DOMS [41][42][43][44]. However, the pain occurring after eccentric exercise is not or only weakly related to the structural muscle damage identi ed in related experiments [20][21][22]. The present study demonstrated that eccentric exercise increased fascia thickness to a considerable degree and this increase correlated with participant's pain sensations. Taken together, our ndings suggest a relevant contribution of the deep fascia to the pathogenesis of DOMS, although a cause-effect relationship is yet to be established.
Changes in pain sensation after eccentric exercise were already evaluated and shown to be related to the fascia in two previous studies [14,16]. Injection of hypertonic sodium solution and electrical stimulation of the connective tissue caused higher intensities and durations of pain than the irritation of the underlying muscle itself [14,16]. However, the mechanisms underlying these observations have not been scrutinized. Solomonow [45] proposed a model describing the degradation of viscoelastic tissue after prolonged high-load exercise. Brie y, it suggests that repeated mechanical strain leads to micro-damage to collagen bers, which, in turn, can trigger in ammation. Indirect signs of collagen breakdown after eccentric exercise have been observed in the form of increased total serum collagen [46, 47] and type IV collagen concentration [48]. Although a cause-effect relationship cannot be derived from our data, it is assumed that the increases in fascia thickness re ect tissue in ammation and/or edema triggered by collagenous micro-failure of the muscular connective tissue, which would be in line with data describing the vulnerability of fascia in muscle injury [10].
We observed an increase in passive muscle tissue displacement after eccentric exercise, but no such change was found in the fascia. Previous studies have revealed that the sliding of the deep fascia against the muscle is a signi cant functional soft tissue property, which, if altered, may induce muscle pain [13]. We expected that damage of the fascia following eccentric exercise might negatively affect both total fascial mobility and tissue gliding. While the former could not be a rmed by our data, muscle displacement increased after eccentric exercise but not after concentric exercise. This was in line with the ndings from Lau et al. [49], who found a signi cant increase (50%) in biceps brachii myotendinous junction displacement during eccentric elbow exor contractions from the rst to the 10th set in the rst bout. After a second bout of the same exercise performed four weeks later, no changes in the displacement were evident. Since DOMS typically occurs after the rst bout of eccentric exercise but not after the second bout, the authors concluded that the change in muscle length affected the magnitude of muscle damage. With regard to our data, taking together both, the increased muscle displacement in the passive movement after eccentric exercise and the constant fascia mobility suggest that the deep fascia was unable to adapt to the higher muscle movement. Yet, as this observation was not correlated with the magnitude of DOMS, future research is warranted to conclusively judge the relevance of an altered muscle-fascia mobility ratio. Some methodological issues need to be discussed. During measurements, no electromyographic recordings were made in order to verify that the movement induced by the isokinetic dynamometer was purely passive. However, this can be assumed with a relatively high degree of certainty as previous studies showed that such continuous passive motion does not provoke muscle activity [34,38]. Another issue relates to the sample size. Our study was explorative in nature and the identi cation of betweencondition effects with only 11 (10 for tissue displacement) participants suggests a relevant implication of fascia morphology in DOMS. However, with regard to insigni cant parameters (i.e. fascia mobility), future studies with larger sample sizes may still observe small-magnitude effects. Finally, it is worth mentioning that a wash-out phase between conditions (concentric and eccentric exercise) was omitted. A carryover effect could thus have in uenced the measurements from a theoretical point of view; however, it is unlikely that the results were largely affected by a carryover effect (e.g., contralateral repeated bout effect) to make the comparison between eccentric and concentric exercise conditions unreasonable.

Conclusions
Eccentric exercise increased fascia thickness, which was associated with the magnitude of DOMS. These ndings support and expand the available evidence suggesting an involvement of the extramuscular connective tissue in the pathogenesis of DOMS. Future studies should (1) clarify the potential association of eccentric loading, connective tissue damage, local in ammation and DOMS, and (2) develop and test speci c exercise paradigms addressing the deep fascia for recovery.   Table   Table 1 Values for fascia thickness, fascia displacement and muscle displacement in the corresponding condition at the different times including all participants. Values are given in mm. The percentage indicate the increase/decrease compared to baseline. (EE = eccentric exercise; CE = concentric exercise, # = mean ± standard deviation, § = median (minimummaximum)).     Changes in pressure pain threshold (PPT) and pain on palpation (visual analogue scale, VAS) pain after exercise. Figure shows parametric (PPT) and non-parametric (VAS) 95% con dence intervals. EE = eccentric exercise, CE = concentric exercise.

Figure 5
Changes in fascia thickness after exercise. Figure shows parametric 95% con dence intervals. EE = eccentric exercise, CE = concentric exercise. Figure 6