# Heterogeneity of Muscle Damage Induced by Electrostimulation: A Multimodal MRI Study

ALEXANDRE FOURÉ¹, GUILLAUME DUHAMEL¹, JENNIFER WEGRZYK¹, HÉLÈNE BOUDINET², JEAN-PIERRE MATTEI¹,³, ARNAUD LE TROTER¹, DAVID BENDAHAN¹, and JULIEN GONDIN¹

¹Aix-Marseille University, Centre National de la Recherche Scientifique (CNRS), Centre de Résonance Magnétique Biologique et Médicale (CRMBM), Unité Mixte de Recherche 7339, Marseille, FRANCE; ²Assistance Publique des Hôpitaux de Marseille (APHM), La Timone Hospital, Imaging Center, Centre d'Exploration Métabolique par Résonance Magnétique (CEMEREM), Marseille, FRANCE; and ³APHM, Sainte Marguerite Hospital, Department of Rheumatology, Marseille, FRANCE

## ABSTRACT

FOURÉ, A., G. DUHAMEL, J. WEGRZYK, H. BOUDINET, J.-P. MATTEI, A. LE TROTER, D. BENDAHAN, and J. GONDIN. Heterogeneity of Muscle Damage Induced by Electrostimulation: A Multimodal MRI Study. *Med. Sci. Sports Exerc.*, Vol. 47, No. 1, pp. 166–175, 2015. **Purpose:** Neuromuscular electrostimulation (NMES) leads to a spatially fixed, synchronous, and superficial motor unit recruitment, which could induce muscle damage. Therefore, the extent of muscle damage and its spatial occurrence were expected to be heterogeneous across and along the *quadriceps femoris* (QF) muscles. The aim of the present study was to characterize muscle spatial heterogeneity in QF damage after a single bout of isometric NMES using multimodal magnetic resonance imaging (MRI). **Methods:** Twenty-five young healthy males participated in this study. MRI investigations consisted of the assessment of muscle volume, transverse relaxation time ($T_2$), and diffusion tensor imaging (DTI) in muscles positioned near the stimulation electrodes (i.e., *vastus lateralis* (VL) and *vastus medialis* (VM)) and muscles located outside the stimulated regions (i.e., *vastus intermedius* and *rectus femoris*). These measurements were performed 6 d before, and 2 d and 4 d (D4) after the NMES session. **Results:** For the muscles placed in direct contact with the stimulation electrodes, volume (VL, +8.5%; VM, +3.8%), $T_2$ (VL, +19.5%; VM, +6.7%) and radial diffusivity ($\lambda_3$) (VL, +7.3%; VM, +3.7%) significantly increased at D4. Whereas MRI parameter changes were larger for VL as compared with those for other QF muscles at D4, homogeneous alterations were found along all QF muscles. **Conclusions:** Isometric NMES induced specific and localized alterations in VL and VM, with heterogeneous damage amplitude among them. Potential effects of unaccustomed intermuscle shear stress during electrically evoked isometric contractions could be a key factor in the spatial occurrence and the extent of damage among QF muscles (especially in VL). The kinetics and extent of MRI changes varied between $T_2$ and diffusion tensor imaging metrics, suggesting the involvement of different physiological processes. **Key Words:** ISOMETRIC NMES CONTRACTION, DIFFUSION TENSOR IMAGING, MRI, TRANSVERSE RELAXATION TIME, QUADRICEPS MUSCLES VOLUME

Neuromuscular electrostimulation (NMES) consists of electrically evoked muscle contractions using surface electrodes placed over the muscle to activate intramuscular nerve branches (14). Although NMES is largely considered a suitable tool for improving, maintaining, or restoring muscle functional capacities (4,10), recent studies have provided evidence for potential deleterious effects. In humans, NMES-induced isometric contractions have been related to decrease in maximal force production and increase in both delayed-onset muscle soreness and creatine kinase (CK) activity (3,15,16). In addition, biopsy sample analyses illustrated major histological alterations such as z-line disruption

and macrophage infiltration (22,23). These damaging effects of isometric NMES have been explained by the specific motor unit recruitment, which is spatially fixed and temporally synchronous, leading to the activation of both slow- and fast-twitch fibers at relatively low force levels (24).

Regarding the spatial selectivity of NMES, it is noteworthy that muscle areas located beneath and close to the stimulation electrodes (i.e., superficial) are primarily recruited at low stimulation intensity, i.e., 5%–10% of maximal voluntary contraction (35). Interestingly, deeper muscle fibers can be activated by increasing the stimulation intensity (1) while the superficial muscle fibers are still depolarized. Considering this peculiar recruitment pattern for NMES-induced isometric contractions, one could reasonably assume that muscle damage would be heterogeneous among and along *quadriceps femoris* (QF) muscles.

Magnetic resonance imaging (MRI) is a powerful noninvasive tool allowing for spatially resolved analysis of muscle tissue (5,7,17). Using transverse relaxation time ($T_2$)-weighted or diffusion tensor imaging (DTI), quantitative MRI indices have been reported to be modified as a result of muscle injury (7,9,26,37). Many studies have observed $T_2$ changes resulting from voluntary eccentric exercise, thereby indicating an

<footnote>Address for correspondence: Alexandre Fouré, Ph.D., Centre de Résonance Magnétique Biologique et Médicale, UMR CNRS 7339, Faculté de Médecine la Timone, 27 Boulevard Jean Moulin, 13385 Marseille, France; E-mail: alexandre.foure@hotmail.fr.
Submitted for publication January 2014.
Accepted for publication May 2014.</footnote>
inflammatory/edematous process (7,11,30). On the contrary, the potential muscle alterations resulting from electrically evoked contractions have been poorly investigated (5-7). Increased $T_2$ values have been recently reported in all four muscles of the QF submitted to electrically evoked lengthening contractions (7). Interestingly, these $T_2$ changes were not uniform across the QF muscle but showed a larger $T_2$ increase in the *vastus medialis* (VM) muscle as compared with that in the *vastus lateralis* (VL) and the *vastus intermedius* (VI) (7). It is noteworthy that these alterations were recorded with stimulation electrodes positioned beneath both VM and VL muscles, thereby indicating that muscle injury can be heterogeneous between the muscles located in direct contact with the stimulation electrodes (7) defined in the present study as "directly activated muscles." In addition, the extent of muscle damage was lower in the proximal slices of QF muscle (i.e., for the muscle areas located far from the stimulation electrodes) than that in the distal ones (5). However, it should be pointed out that the corresponding muscle alterations resulted from electrically evoked lengthening contractions. On that basis, different muscle length changes occurring both among and along muscles can be viewed as a confounding factor involved in the previously mentioned muscle damage heterogeneity. Indeed, voluntary eccentric exercise has been related to much higher $T_2$ increase in the *rectus femoris* (RF) muscle as compared that in the *vastii* muscles (30). Considering that NMES resistance exercise is typically delivered under isometric conditions, the heterogeneity and extent of the corresponding damage should be specifically investigated among and along individual muscles for this particular stimulation modality.

Combined measurements of muscle volume, $T_2$ values, and DTI metrics have been recognized as relevant indices of edema, inflammation processes (18,25,32), and cellular membrane disruption (9,13,26), thus being well suited to shed light on the specific localization of muscle damage among and along QF muscles. In addition, changes in DTI metrics have been reported in the early phase of muscle damage as compared with changes in muscle volume and $T_2$ values (13). On that basis, DTI metrics could provide important clues on subtle alterations of muscle microstructure during damaging exercise.

The purpose of the present study was to quantify and localize isometric NMES-induced muscle damage using multimodal MRI within the QF muscles. We hypothesized that larger change in muscle volume, $T_2$, and DTI metrics would occur for the directly activated muscles (i.e., VL and VM) as compared with the muscles located far from the stimulation electrodes (i.e., VI and RF, defined in the present study as "non-directly activated muscles"). In addition, larger alterations could be expected within areas located beneath the stimulation electrodes.

## METHODS

**Subjects.** Twenty-five healthy men (age, 22 (2) yr; height, 178 (6) cm; body mass, 68 (7) kg) volunteered to participate in this study. None of them were engaged in any

training or exercise programs. Subjects were instructed to avoid any intensive and nonfamiliar physical activities throughout the duration of the protocol. Volunteers were fully informed about the nature and the aim of the study and gave their written informed consent to participate in this study. The study was approved by the Local Human Research Ethics Committee Sud Mediterranée V (2012-04 A00449-34) and was conducted in conformity with the Declaration of Helsinki. Subjects were asked to keep their diet habits and to limit their alcohol consumption throughout the study period. They were instructed to avoid consuming caffeine and smoking before the experiments. The consumption of medicines was prohibited during 7 d before and 5 d after the NMES session.

**Study design.** Assessments of muscle force, soreness, and plasma CK activity were performed just before the NMES session, immediately after exercise-induced muscle damage, and 1 (D1), 2 (D2), 3 (D3), and 4 d (D4) after the NMES session. All testing sessions were performed at the same time of day. Three identical MRI sessions were performed 6 d before NMES exercise (D-6), at D2, and D4 to assess QF muscles volume, $T_2$, and DTI metrics.

**NMES session.** The subject was seated on a specific chair (Multi-Form, La Roque d'Anthéron, France) customized with a force sensor. Hip and ankle joints were secured by adjustable lap belts to hold hip and knee angles at approximately 90° and 100°, respectively (0° corresponds to the full extension). Both legs were stimulated simultaneously using three electrodes placed over the thigh skin: a 5 × 10 cm on the proximal part of the thigh and two 5 × 5 cm on the VL and VM muscle bellies detected by manual muscle palpation during submaximal voluntary isometric contractions. Biphasic symmetric rectangular pulses were delivered at a frequency of 100 Hz with a pulse duration of 400 µs (40 contractions, a 5-s on/35-s off ratio throughout the NMES exercise) using a portable battery-powered stimulator (Compex® Performance, Djo Global, France). Stimulation intensity was gradually and similarly increased in both the VL and VM muscles to a level of maximum tolerance throughout the NMES protocol. The exact electrode positions were marked on the skin surface with permanent ink immediately after the NMES session.

Force signal was recorded and sampled at 1000 Hz using a PowerLab system and Labchart software (ADInstruments, Colorado Springs, CO) and stored for analysis with a customized MATLAB program (version 7.11; Mathworks, Natick, MA). For each NMES-evoked contraction, the peak force was measured and averaged over the forty contractions.

**Maximal voluntary isometric contraction force.** The subject was seated on a chair with the knee flexed at approximately 100° and performed a 5- to 10-min warm-up including a set of submaximal knee extensions under isometric conditions for both legs simultaneously (i.e., bilateral contractions). Subjects were instructed to perform three unilateral isometric maximal voluntary contractions (MVC) with the right leg. The MVC trials were separated by a resting period of at least 30 s and quantified as the highest value of the three trials.
**Muscle soreness.** The level of muscle soreness was assessed at rest, during squat isometric contraction, passive stretching, and by palpation of VL and VM on the basis of a visual analog scale with a 100-mm horizontal line with “no pain” on one end (0 mm) and “extremely painful” on the other (100 mm). Squat movement was performed from standing position to a knee flexion angle of 90° with a constant movement velocity (i.e., 2 s for movement from standing position). Passive stretching consisted in knee joint passive flexion. During palpation of VL and VM, the knee was flexed at approximately 100°. Soreness was assessed using the mean value obtained from the five measures (i.e., at rest, isometric squat, passive stretching, and VL and VM palpation).

**Plasma CK activity.** Blood was drawn from the forearm vein of each subject (5 mL) after a standardized procedure. Plasma was immediately separated after blood centrifugation (15 min at 2500 rpm). Plasma CK activity was determined enzymatically at 37°C using a chemistry analyzer (UniCel DxC 800 Synchron Clinical System; Beckman Coulter, Inc., Brea, CA).

**MRI.** Subjects were positioned supine, with the right leg centered in a 1.5-T super-conducting magnet (MAGNETOM Avanto; Siemens AG, Healthcare Sector, Erlangen, Germany). A flexible surface six-channel body coil (Siemens AG, Healthcare Sector, Erlangen, Germany) was placed around the right QF muscles. QF muscles volume was determined from high-resolution T₁-weighted images (20 slices; field of view (FOV), 220 × 220 mm; matrix = 576 × 576; time repetition (TR) = 549 ms; time echo (TE) = 13 ms; number of repetitions (N<sub>EX</sub>) = 1; slice thickness = 6 mm; gap between slices = 6 mm; acquisition time = 5 min 18 s). T₂-weighted images were acquired with a segmented (15 segments) echo planar imaging sequence with TE of 15, 25, 35, 45, and 55 ms. Other parameters were as follows: 20 slices; FOV = 220 × 220 mm; matrix = 192 × 192; TR = 4800 ms; N<sub>EX</sub> = 1; slice thickness = 6 mm; gap between slices = 6 mm, short time inversion

recovery (STIR) for fat saturation; acquisition time = 5 min 10 s. Diffusion-weighted images were acquired with a modified Stejskal-Tanner single-shot spin-echo planar imaging sequence (27,33) and the following parameters: 20 slices; FOV = 220 × 220 mm; matrix = 96 × 96; TR = 5000 ms; TE = 53 ms; N<sub>EX</sub> = 6; slice thickness = 6 mm; gap between slices = 6 mm, 12 directions; 2 b-value (0 and 500 s·mm<sup>-2</sup>); bandwidth = 2264 Hz/Px; acquisition time = 6 min 57 s.

The most distal slice (i.e., slice 0) (Fig. 1) was always acquired at approximately 20 mm (i.e., 5% of the thigh length measured for each subject) up the proximal border of the patella. The stimulation electrodes were carefully localized using oil capsules stuck on the skin surface and were observable on T₁-weighted images (circular hypersignals) (Fig. 1). Oil capsules were observable on slices 2 (1) and 13 (2) for VM and VL, respectively.

Images were analyzed with FSLview (FMRIB, Oxford, MS). Regions of interest (ROI) were drawn in each slice by manually tracing the border of the anatomic cross-sectional area of each QF muscle. Using the truncated cone formula (28), the volume of QF muscles was calculated by summing the areas of all slices while taking into account the slice thickness and the gaps between slices. T₂ maps were generated by fitting on a pixel-by-pixel basis the logarithm of the data to the equation 1:

$$ \ln[S(TE)] = \ln(S_0) - (TE/T_2) \quad [1] $$

where S(TE) is the signal at time equal to TE and S₀ is the equilibrium magnetization. Thereafter, T₂ maps were resized to T₁-weighted image definition. ROI initially drawn on T₁-weighted images were used to analyze T₂ maps and determine a mean value of T₂ for VL, VM, VI, and RF.

The DTI reconstruction was performed with the manufacturer software (syngo MR; Siemens AG, Healthcare Sector, Erlangen, Germany). ROI were specifically drawn on diffusion-weighted images and applied on the quantitative maps to calculate DTI metrics (longitudinal diffusivity

<figure><img src="image_1.png" /><figcaption>FIGURE 1—Representative sagittal image of the thigh (left), with each grid line indicating the location of the 20 axial slices. A proximal and a distal slice are displayed (right) and correspond to the placement of stimulation electrodes on the VL and VM, respectively. The stimulation electrodes were localized by using oil capsules stuck on the skin surface and observable on T₁-weighted images (round hypersignals).</figcaption></figure>
($\lambda_1$) and radial diffusivities ($\lambda_2$ and $\lambda_3$)) for each muscle on each slice. Diffusivities were used to calculate the mean diffusivity (MD) and the fractional anisotropy (FA) according to equations 2 and 3 (37):

$$MD = (\lambda_1 + \lambda_2 + \lambda_3)/3 \quad [2]$$

$$FA = (\sqrt{3}/\sqrt{2}) \times \left( \frac{\sqrt{(\lambda_1 - \text{MD})^2 + (\lambda_2 - \text{MD})^2 + (\lambda_3 - \text{MD})^2}}{\sqrt{\lambda_1^2 + \lambda_2^2 + \lambda_3^2}} \right) \quad [3]$$

**Reproducibility of measurements.** Reproducibility of the MRI parameters (i.e., volume, $T_2$, and DTI metrics) was assessed in a pilot study conducted on seven subjects (three women and four men, 32 (7) yr, 169 (9) cm, 64 (11) kg). Two repeated testing sessions, separated by a 2-d resting period, were performed at the same time of the day. Intraclass correlation coefficients (2, 1), the SEM, and coefficient of variation (CV) were calculated and are presented in Table 1. These results support the reliability of the measurements performed in the present study, especially with CV lower than 4.9% for all parameters.

**Statistics.** After having tested the data distribution, parametric statistical tests were performed using Statistica software (Statsoft, Tulsa, OK). One-way repeated-measures ANOVA were performed to assess changes in MVC, muscle soreness, and plasma CK. Two-factor repeated-measures ANOVA

(muscle × time and slice × time for each muscle) were used to assess changes in muscle volume, $T_2$ values, and DTI metrics among and along the QF muscles. A Tukey HSD *post hoc* analysis was performed when appropriate. The level of significance was set to $P < 0.05$. Results are reported as mean (SD).

## RESULTS

### NMES Session

Stimulation intensity was gradually increased throughout the NMES session from 33 (6) mA and reached 65 (16) mA at the 40th contraction. The averaged bilateral peak force evoked by NMES was 255 (59) N, corresponding to a relative force of 32% (7%) MVC. Electrically evoked force increased in the first 15 contractions and remained constant after the 20th contraction despite the continuous increase in stimulation intensity.

### MVC Force

Unilateral MVC was significantly decreased by 28% immediately after NMES (from 327 (65) to 233 (52) N, $P < 0.05$). Although a significant recovery occurred during the post-NMES period ($P < 0.05$), the corresponding MVC was still lower than the baseline value at D1 (245 (60) N, i.e., 75% MVC), D2 (251 (66) N, i.e., 78% MVC), D3 (255 (71) N, i.e., 79% MVC), and D4 (265 (70) N, i.e., 82% MVC).

<table><caption>TABLE 1. Day-to-day reliability for MRI parameters.</caption><thead><tr><th></th><th></th><th>Test 1</th><th>Test 2</th><th>SEM</th><th>CV (%)</th><th>ICC</th></tr></thead><tbody><tr><td rowspan="4">Muscle volume (cm³)</td><td>VL</td><td>500 (147)</td><td>509 (160)</td><td>17</td><td>2.8</td><td>0.99</td></tr><tr><td>VM</td><td>476 (111)</td><td>476 (110)</td><td>7</td><td>1.4</td><td>1.00</td></tr><tr><td>VI</td><td>501 (102)</td><td>501 (113)</td><td>9</td><td>1.9</td><td>0.99</td></tr><tr><td>RF</td><td>151 (43)</td><td>151 (45)</td><td>3</td><td>2.1</td><td>1.00</td></tr><tr><td rowspan="4">T₂ (ms)</td><td>QF</td><td>1627 (300)</td><td>1638 (329)</td><td>26</td><td>1.4</td><td>0.99</td></tr><tr><td>VL</td><td>32.4 (0.5)</td><td>32.2 (0.7)</td><td>0.3</td><td>0.9</td><td>0.79</td></tr><tr><td>VM</td><td>32.7 (0.5)</td><td>32.5 (0.6)</td><td>0.2</td><td>0.6</td><td>0.90</td></tr><tr><td>VI</td><td>33.3 (0.6)</td><td>32.8 (0.6)</td><td>0.3</td><td>0.8</td><td>0.83</td></tr><tr><td rowspan="4">λ₁ (10⁻³ mm²·s⁻¹)</td><td>RF</td><td>31.5 (0.4)</td><td>31.3 (0.9)</td><td>0.5</td><td>1.6</td><td>0.50</td></tr><tr><td>QF</td><td>32.5 (0.5)</td><td>32.2 (0.7)</td><td>0.3</td><td>0.8</td><td>0.82</td></tr><tr><td>VL</td><td>2.20 (0.13)</td><td>2.09 (0.06)</td><td>0.09</td><td>4.2</td><td>0.17</td></tr><tr><td>VM</td><td>2.11 (0.06)</td><td>2.07 (0.07)</td><td>0.05</td><td>1.9</td><td>0.35</td></tr><tr><td rowspan="4">λ₂ (10⁻³ mm²·s⁻¹)</td><td>VI</td><td>2.14 (0.06)</td><td>2.09 (0.06)</td><td>0.05</td><td>2.5</td><td>0.26</td></tr><tr><td>RF</td><td>2.24 (0.06)</td><td>2.17 (0.07)</td><td>0.04</td><td>2.5</td><td>0.68</td></tr><tr><td>QF</td><td>2.17 (0.06)</td><td>2.10 (0.06)</td><td>0.05</td><td>2.3</td><td>0.36</td></tr><tr><td>VL</td><td>1.75 (0.11)</td><td>1.65 (0.04)</td><td>0.08</td><td>4.8</td><td>0.03</td></tr><tr><td rowspan="4">λ₃ (10⁻³ mm²·s⁻¹)</td><td>VM</td><td>1.68 (0.05)</td><td>1.64 (0.05)</td><td>0.04</td><td>2.3</td><td>0.44</td></tr><tr><td>VI</td><td>1.70 (0.07)</td><td>1.65 (0.05)</td><td>0.04</td><td>2.6</td><td>0.49</td></tr><tr><td>RF</td><td>1.77 (0.06)</td><td>1.70 (0.07)</td><td>0.03</td><td>1.6</td><td>0.84</td></tr><tr><td>QF</td><td>1.72 (0.06)</td><td>1.66 (0.05)</td><td>0.04</td><td>2.5</td><td>0.44</td></tr><tr><td rowspan="4">MD (10⁻³ mm²·s⁻¹)</td><td>VL</td><td>1.42 (0.05)</td><td>1.37 (0.04)</td><td>0.04</td><td>3.2</td><td>0.01</td></tr><tr><td>VM</td><td>1.38 (0.04)</td><td>1.37 (0.04)</td><td>0.02</td><td>1.7</td><td>0.67</td></tr><tr><td>VI</td><td>1.38 (0.05)</td><td>1.36 (0.04)</td><td>0.03</td><td>1.9</td><td>0.70</td></tr><tr><td>RF</td><td>1.41 (0.04)</td><td>1.37 (0.05)</td><td>0.02</td><td>1.8</td><td>0.73</td></tr><tr><td rowspan="4">FA</td><td>QF</td><td>1.40 (0.04)</td><td>1.36 (0.04)</td><td>0.02</td><td>1.5</td><td>0.67</td></tr><tr><td>VL</td><td>1.79 (0.09)</td><td>1.70 (0.04)</td><td>0.07</td><td>4.1</td><td>0.03</td></tr><tr><td>VM</td><td>1.72 (0.05)</td><td>1.69 (0.05)</td><td>0.04</td><td>2.2</td><td>0.45</td></tr><tr><td>VI</td><td>1.74 (0.06)</td><td>1.70 (0.05)</td><td>0.04</td><td>2.3</td><td>0.48</td></tr><tr><td rowspan="4">MD (10⁻³ mm²·s⁻¹)</td><td>RF</td><td>1.81 (0.05)</td><td>1.75 (0.06)</td><td>0.03</td><td>1.5</td><td>0.78</td></tr><tr><td>QF</td><td>1.76 (0.05)</td><td>1.71 (0.05)</td><td>0.04</td><td>2.2</td><td>0.44</td></tr><tr><td>VL</td><td>0.22 (0.01)</td><td>0.21 (0.01)</td><td>0.01</td><td>3.1</td><td>0.43</td></tr><tr><td>VM</td><td>0.21 (0.01)</td><td>0.21 (0.01)</td><td>0.01</td><td>3.0</td><td>0.64</td></tr><tr><td rowspan="4">FA</td><td>VI</td><td>0.22 (0.01)</td><td>0.22 (0.01)</td><td>0.01</td><td>2.8</td><td>0.52</td></tr><tr><td>RF</td><td>0.23 (0.02)</td><td>0.23 (0.01)</td><td>0.01</td><td>3.6</td><td>0.57</td></tr><tr><td>QF</td><td>0.22 (0.01)</td><td>0.22 (0.01)</td><td>0.01</td><td>2.0</td><td>0.46</td></tr></tbody></table>

ICC, intraclass correlation coefficient; $\lambda_1/\lambda_2/\lambda_3$, first, second, and third eigenvalues from diffusion tensor.
## Muscle Soreness

Muscle soreness significantly increased at D1 (3 (4) mm), peaked at D2 (14 (12) mm), and progressively decreased at D3 (12 (11) mm) and D4 (7 (8) mm) but was still higher than the baseline values (P < 0.05).

## Plasma CK Activity

Plasma CK activity significantly increased (P < 0.05) at D2 (1848 (2191) IU·L⁻¹) and D3 (7227 (7499) IU·L⁻¹) and reached a peak at D4, corresponding to 70-fold increase (from 171 (135) IU·L⁻¹ at baseline to 12,460 (17,206) IU·L⁻¹ at D4).

## MRI Parameters

**Volume.** A significant increase in QF muscle volume was measured at D2 (+3.6%, P < 0.05) and D4 (+4.7%, P < 0.05) (Table 2). A significant muscle-time interaction (P < 0.05) was found, so that directly activated muscles showed significant increase (P < 0.05) in muscle volume at D2 (+5.4% for VL and +4.2% for VM) and at D4 (+8.5% for VL and +3.8% for VM). In addition, VL muscle volume increased by 3.0% from D2 to D4 (P < 0.05). On the contrary, no significant change occurred in the RF muscle (P > 0.05)

and a slight but significant increase in VI muscle volume was observed at D4 as compared with that in baseline (+2.2%) (Table 2). Overall, changes in VL muscle volume were significantly larger as compared with those in other QF muscles (P < 0.05)

No significant slice-time interaction was found (P > 0.05), thereby indicating that changes in volume were homogeneous along QF muscles (Fig. 2).

**T₂.** T₂ values significantly increased in the QF muscles at D2 (+5.9%, P < 0.05) and D4 (+8.6%, P < 0.05). Similar to the muscle volume, T₂ changes were heterogeneous between directly and non-directly activated muscles. Indeed, a significant interaction between muscle and time was found (P < 0.05). T₂ changes were only significant in directly activated muscles at D2 (+10.3% for the VL and +5.7% for the VM), with larger increase in the VL at D4 (+19.5%) as compared with that in the VM (+6.7%) (Table 2). No T₂ change was observed in non-directly activated muscles at D2 and D4 (P > 0.05).

No significant slice-time interaction was found, indicating similar T₂ changes along the QF muscles (Fig. 3).

**DTI metrics.** A significant muscle-time interaction was only found in λ₃ and FA (P < 0.05) (Table 2). λ₃ increased

<table><caption>TABLE 2. MRI parameters assessed at 6 d before (D-6) and 2 (D2) and 4 d (D4) after NMES-induced muscle damage.</caption><thead><tr><th></th><th></th><th>D-6</th><th>D2</th><th>D4</th></tr></thead><tbody><tr><td rowspan="5">Muscle volume (cm³)</td><td>VL</td><td>544 (103)<sup>S</sup></td><td>572 (102)<sup>*</sup>,<sup>S</sup></td><td>589 (108)<sup>*</sup>,<sup>#</sup>,<sup>S,ℓ,§</sup></td></tr><tr><td>VM</td><td>505 (78)<sup>S</sup></td><td>525 (76)<sup>*</sup>,<sup>S</sup></td><td>524 (77)<sup>*</sup>,<sup>S</sup></td></tr><tr><td>VI</td><td>569 (100)<sup>S</sup></td><td>579 (96)<sup>S</sup></td><td>581 (100)<sup>*</sup>,<sup>S</sup></td></tr><tr><td>RF</td><td>168 (45)</td><td>171 (43)</td><td>172 (43)</td></tr><tr><td><strong>QF</strong></td><td><strong>1786 (298)</strong></td><td><strong>1846 (283)<sup>*</sup></strong></td><td><strong>1866 (293)<sup>*</sup></strong></td></tr><tr><td rowspan="5">T₂ (ms)</td><td>VL</td><td>32.5 (0.9)</td><td>35.8 (1.9)<sup>*</sup>,<sup>S</sup></td><td>38.8 (5.0)<sup>*</sup>,<sup>#</sup>,<sup>S,ℓ,§</sup></td></tr><tr><td>VM</td><td>32.9 (0.8)</td><td>34.8 (1.1)<sup>*</sup>,<sup>S,ℓ</sup></td><td>35.1 (1.6)<sup>*</sup>,<sup>S,ℓ</sup></td></tr><tr><td>VI</td><td>33.1 (0.9)</td><td>34.3 (1.0)</td><td>34.5 (1.6)</td></tr><tr><td>RF</td><td>31.7 (0.9)</td><td>32.7 (1.1)</td><td>32.7 (1.3)</td></tr><tr><td><strong>QF</strong></td><td><strong>32.6 (0.8)</strong></td><td><strong>34.5 (1.0)<sup>*</sup></strong></td><td><strong>35.4 (2.0)<sup>*</sup></strong></td></tr><tr><td rowspan="5">λ₁ (10⁻³·mm²·s⁻¹)</td><td>VL</td><td>2.14 (0.14)</td><td>2.18 (0.11)</td><td>2.16 (0.05)</td></tr><tr><td>VM</td><td>2.09 (0.04)</td><td>2.13 (0.05)</td><td>2.14 (0.03)</td></tr><tr><td>VI</td><td>2.12 (0.07)</td><td>2.16 (0.08)</td><td>2.15 (0.05)</td></tr><tr><td>RF</td><td>2.16 (0.05)</td><td>2.18 (0.07)</td><td>2.18 (0.07)</td></tr><tr><td><strong>QF</strong></td><td><strong>2.12 (0.06)</strong></td><td><strong>2.16 (0.06)</strong></td><td><strong>2.17 (0.05)</strong></td></tr><tr><td rowspan="5">λ₂ (10⁻³·mm²·s⁻¹)</td><td>VL</td><td>1.66 (0.06)</td><td>1.70 (0.08)</td><td>1.70 (0.06)</td></tr><tr><td>VM</td><td>1.66 (0.03)</td><td>1.69 (0.05)</td><td>1.69 (0.03)</td></tr><tr><td>VI</td><td>1.67 (0.05)</td><td>1.69 (0.05)</td><td>1.69 (0.04)</td></tr><tr><td>RF</td><td>1.71 (0.04)</td><td>1.75 (0.06)</td><td>1.73 (0.06)</td></tr><tr><td><strong>QF</strong></td><td><strong>1.66 (0.04)</strong></td><td><strong>1.70 (0.05)</strong></td><td><strong>1.70 (0.04)</strong></td></tr><tr><td rowspan="5">λ₃ (10⁻³·mm²·s⁻¹)</td><td>VL</td><td>1.36 (0.05)</td><td>1.42 (0.05)<sup>*</sup>,<sup>S</sup></td><td>1.44 (0.06)<sup>*</sup>,<sup>#</sup>,<sup>S,ℓ,§</sup></td></tr><tr><td>VM</td><td>1.39 (0.03)</td><td>1.43 (0.03)<sup>*</sup>,<sup>S</sup></td><td>1.43 (0.03)<sup>*</sup>,<sup>S</sup></td></tr><tr><td>VI</td><td>1.38 (0.03)</td><td>1.40 (0.03)<sup>*</sup>,<sup>S</sup></td><td>1.40 (0.03)<sup>*</sup>,<sup>S</sup></td></tr><tr><td>RF</td><td>1.41 (0.03)</td><td>1.43 (0.04)</td><td>1.43 (0.04)</td></tr><tr><td><strong>QF</strong></td><td><strong>1.37 (0.04)</strong></td><td><strong>1.41 (0.03)<sup>*</sup></strong></td><td><strong>1.42 (0.04)<sup>*</sup></strong></td></tr><tr><td rowspan="5">MD (10⁻³·mm²·s⁻¹)</td><td>VL</td><td>1.72 (0.07)</td><td>1.77 (0.08)</td><td>1.77 (0.05)</td></tr><tr><td>VM</td><td>1.71 (0.03)</td><td>1.75 (0.04)</td><td>1.76 (0.03)</td></tr><tr><td>VI</td><td>1.72 (0.05)</td><td>1.75 (0.05)</td><td>1.75 (0.03)</td></tr><tr><td>RF</td><td>1.76 (0.03)</td><td>1.79 (0.05)</td><td>1.78 (0.05)</td></tr><tr><td><strong>QF</strong></td><td><strong>1.72 (0.04)</strong></td><td><strong>1.76 (0.04)</strong></td><td><strong>1.76 (0.04)</strong></td></tr><tr><td rowspan="5">FA</td><td>VL</td><td>0.22 (0.02)</td><td>0.22 (0.01)</td><td>0.21 (0.02)<sup>*</sup>,<sup>#</sup>,<sup>S,ℓ,§</sup></td></tr><tr><td>VM</td><td>0.21 (0.01)</td><td>0.20 (0.01)</td><td>0.21 (0.01)</td></tr><tr><td>VI</td><td>0.22 (0.01)</td><td>0.22 (0.02)</td><td>0.22 (0.01)</td></tr><tr><td>RF</td><td>0.22 (0.01)</td><td>0.21 (0.01)</td><td>0.22 (0.01)</td></tr><tr><td><strong>QF</strong></td><td><strong>0.22 (0.01)</strong></td><td><strong>0.22 (0.01)</strong></td><td><strong>0.22 (0.01)</strong></td></tr></tbody></table>

λ₁/λ₂/λ₃, first, second and third eigenvalues from diffusion tensor. Bold values are related to quadriceps femoris (QF) muscle group.

*Significantly different from baseline (P < 0.05).

#Significantly different from D2 (P < 0.05).

§Significantly different from RF (P < 0.05).

‡Significantly different from VI (P < 0.05).

§Significantly different from VM (P < 0.05).
<figure><img src="image_2.png" /><figcaption>FIGURE 2—Changes in muscle volume measured over the 20 slices in the VL (A), VM (B), VI (C), and RF (D) muscles 2 d (empty diamonds and solid line) and 4 d (full circles and dotted line) after the NMES session.</figcaption></figure>

significantly not only for directly activated muscles (+4.7% and +3.4% at D2 and +7.3% and +3.7% at D4 for the VL and VM, respectively) but also for the non-directly activated VI muscle (+2.1% and +2.3% at D2 and D4, respectively). A

significant increase between D2 and D4 was also found in $\lambda_3$ for VL ($P < 0.05$), so that changes in $\lambda_3$ were larger for the VL as compared with those for other QF muscles at D4 ($P < 0.05$).

<figure><img src="image_3.png" /><figcaption>FIGURE 3—Changes in muscle $T_2$ on the 20 slices and determined 2 d (D2, empty diamonds and solid line) and 4 d (D4, full circles and dotted line) after NMES session for VL (A), VM (B), VI (C), and RF (D) muscles. For the three MRI investigations (D-6, D2, and D4), $T_2$ maps superimposed on $T_1$-weighted images are displayed of a representative subject (E) for three slices, corresponding to distal (slice 3), middle (slice 10), and proximal (slice 14) areas of the QF.</figcaption></figure>
Moreover, FA significantly decreased in the VL at D4 (−7.0%) whereas no change occurred in the three other QF muscles. All DTI metrics remained unaffected in the RF muscle (Table 2). In addition, no significant slice-time interaction was found in $\lambda_3$ (Fig. 4) of the QF muscles and in FA of VL.

DISCUSSION

The major findings of the present study were that MRI parameters were largely and heterogeneously modified as a result of a single bout of isometric NMES, thereby clearly illustrating major muscle alterations. We showed that isometric NMES-induced muscle damage was heterogeneous between directly (i.e., VL and VM) and non-directly (i.e., VI and RF) activated muscles but also among the VL and VM. On the contrary, the extent of alteration was homogeneous along the QF muscle. Our results also indicated that the time course of $T_2$ values and DTI metrics varied, thereby likely reflecting different physiological processes.

MVC force loss is currently considered the most reliable indicator of muscle injury (36). In our study, the immediate and prolonged MVC force losses (i.e., approximately 30% and approximately 20% at D4, respectively) were in agreement with those reported in previous NMES studies (3,16,34). In addition, the time course of other indirect markers of muscle damage, including delayed-onset muscle soreness and CK activity (i.e., peaking at D2 and D4, respectively), was

similar to that in previous studies assessing the effects of isometric NMES (3,15). Despite the large changes regarding force production, enzymatic activities, and MRI parameters, the level of muscle soreness reported by our subjects was relatively low as compared with what has been reported in previous NMES studies (e.g., 2). It has been well acknowledged that muscle soreness is a subjective variable, which does not reflect the magnitude of exercise-induced muscle damage (29). In our study, almost all the subjects were recruited from the Medical School of Aix—Marseille University for whom the rating “extremely painful” would have corresponded to a pain sensation experienced by patients with cancer during the terminal phase. Our study provides further evidence that muscle soreness is a poor indicator of the magnitude of muscle damage, as previously suggested (29,36). In the present study, we combined multimodal MRI measurements to assess muscle damage in knee extensors more specifically and objectively. MRI parameter values were in the same range as those obtained in previous studies investigating muscle injury (7,9,30,38).

We found that both QF volume and $T_2$ values were respectively increased by approximately 5% and approximately 9% as a result of isometric NMES. These changes were lower than the respective increase of 10% and 16% in QF volume and $T_2$ values recently reported for electrically evoked eccentric contractions (7), thereby highlighting the influence of muscle length changes on the magnitude of muscle injury (19). Moreover, although DTI metrics have never been assessed after NMES-induced muscle damage,

<figure><img src="image_4.png" /><figcaption>FIGURE 4—Changes in muscle $\lambda_3$ on the 20 slices and determined 2 d (D2, empty diamonds and solid line) and 4 d (D4, full circles and dotted line) after NMES session for VL (A), VM (B), VI (C), and RF (D) muscles. $\lambda_3$ maps superimposed on $T_1$-weighted images are displayed of a representative subject (E), considering the three MRI investigations (D-6, D2, and D4) and three slices corresponding to proximal (slice 3), midthigh (slice 10), and proximal (slice 14) areas of the QF.</figcaption></figure>
Cermak et al. (9) found a significant increase in $\lambda_3$ of the whole QF 1 d after voluntary eccentric actions of the knee extensors, which is in agreement with the $\lambda_3$ increase found 2 d after the isometric NMES exercise in our study.

We showed that MRI parameters were heterogeneously affected after isometric NMES contractions, similar to what has been recently described after electrically evoked eccentric contractions (5–7). In the days after NMES-induced muscle damage, a significant increase in muscle volume and $T_2$ occurred exclusively for the directly activated muscles, whereas no or slight changes were observed in non-directly activated muscles. In additional experiments conducted among four men (25 (2) yr, 178 (3) cm, 75 (7) kg), we identified activation areas using $T_2$ mapping recorded before and immediately after the 40 isometric NMES contractions (delay of 215 (5) s between the last NMES isometric contraction and $T_2$ acquisition). On the basis of $T_2$ changes, we showed similar activation for the VL ($\Delta T_2$ = 16% (5%)) and the VM ($\Delta T_2$ = 14% (6%)) muscles within slices, corresponding to the stimulation electrodes position. On the contrary, the deeper VI muscle was poorly activated ($\Delta T_2$ = 1% (1%)). Muscle damage is not linked to the force level per se but, rather, to the active strain supported by the muscle (19), which is in accordance with the minimal activation of VI and the low RF length during the isometric NMES protocol performed in the present study.

In contrast to changes in volume and $T_2$ values, variations in DTI metrics did not exclusively occur in the directly activated muscles. Indeed, a significant increase in radial diffusivity of water molecules (i.e., $\lambda_3$) was shown within the three *vastii* indicating muscle microstructural alterations (20), although VI was less activated during isometric NMES contractions as compared with the VL and VM. Interestingly, the $\lambda_3$ increase in VI occurred before any change in muscle volume, which is consistent with both human (37) and animal studies (13). Moreover, the additional increase in VL volume and $T_2$ occurring between D2 and D4 was also accompanied by a significant decrease in FA at D4, thereby highlighting an inhomogeneous alteration among the directly activated muscles.

The stimulation intensity was similarly increased in both the VM and VL muscles during the NMES session, and, on the basis of $T_2$ changes measured in additional experiments, the corresponding activation of the directly activated muscles immediately after NMES exercise was similar. Black et al. (7) found a larger $T_2$ change in the VM as compared with that in other QF muscles 3 d after NMES eccentric contractions. Surprisingly, we found that alterations were more marked in the VL, suggesting that magnitude of muscle damage was not linked to the level of muscular activation per se (19) but rather to an unaccustomed pattern of activation in VL. Indeed, on the basis of M-wave analyses, activation according to the size principle (i.e., an orderly recruitment from slow to fast motor units) was demonstrated during electrically isometric evoked contractions for the VM (31). On the contrary, isometric NMES leads to a random

recruitment of motor units in VL (31). On that basis, the heterogeneous muscle alterations occurring across the directly activated muscles might be related to a different activation pattern, leading to an unaccustomed stress applied on VL during NMES-induced muscle contraction. Moreover, the anatomical position and/or the proximity with other muscles within QF muscle group (12) could also contribute to the higher alterations found in VL. Indeed, the significant increase of both volume and $\lambda_3$ in VI muscle could be related to a shear stress applied between VL and VI during isometric NMES contractions. This hypothesis is reinforced by visual inspection of the $T_2$ maps (Fig. 3-E) showing that muscle alterations were larger in deeper VL muscle areas, i.e., those in close vicinity to the VI muscle areas. This could be due to potential frictions between an active muscle (i.e., VL) and a coplanar non-directly activated muscle (i.e., VI). This assumption is actually supported by results related to the potential lateral force transmission through the connective tissue linkages (e.g., 21) and those illustrating different displacements among the synergistic muscles during isometric contraction (8). In addition, the contact area between the VL and VI muscles is higher than the corresponding area between the VM and VI muscles, especially at the level of the slice where the stimulation electrodes were placed. It is noteworthy that VL and VI are in contact in all slices and VL was activated on its full length whereas VM was mainly activated in distal areas where potential frictions between VM and VI are restricted. On that basis, shear stress between VL and VI muscles could be considered as the main accounting factor of muscle damage, which is in line with the recent studies showing connective tissue alterations and extracellular matrix deadhesion after isometric NMES exercise within the muscle (22,23). Moreover, a recruitment of deeper nerve branches due to the increased stimulation intensity during NMES exercise (1) combined with a neuromuscular propagation failure (39) in superficial muscle fibers could lead to the activation and *in fine* alteration of deep VL muscle fibers. Further studies are needed to accurately quantify the spatial distribution of alterations within each muscle to discriminate potential causes of muscle damage induced by a single session of NMES.

For all MRI-investigated parameters, we found homogeneous changes along the four QF muscles. Our findings do not support the results from Black et al. (7) illustrating reduced muscle damage in the proximal part of the QF muscle after electrically evoked eccentric contractions (5), i.e., for the muscle areas located far from the stimulation electrodes. It should be pointed out that we focused our MRI measurements on stimulated muscle areas and did not investigate the most proximal part of the QF muscles previously shown to be less activated (5) during electrically evoked eccentric contractions. However, the latter study found homogeneous alterations along the QF muscles, considering distal and central areas, which is in accordance with the results obtained in the present study. Thus, the homogeneous alteration occurring along the full length of muscles reinforces
the hypothesis of a global alteration of the whole muscle due to peripheral stress between muscles. Overall, the accurate localization of muscle damage among and along the QF muscles is of utmost interest for improving our understanding of the mechanisms underlying muscle damage and for determination of specific muscle area for biopsy sampling (22,23).

In conclusion, we demonstrated that a single bout of isometric NMES leads to major muscle fiber alterations that are heterogeneous between the directly activated muscles (i.e., VL and VM) as compared with other QF muscles but also among VL and VM. We suggested that shear stress between an active muscle (i.e., VL) and a less activated muscle (i.e., VI) could be an important accounting factor of muscle damage. However, further studies are needed to identify and quantify the heterogeneity of alterations within each muscle that would provide additional information on the potential causes of observed muscle damage. In addition, the kinetics and extent of changes varied between T₂ and DTI metrics, indicating that the corresponding indices likely reflect different physiological

processes and highlighting the relevance of multimodal MRI for monitoring in vivo skeletal muscle injury.

The authors thank the Centre de Pharmacologie Clinique et d'Evaluations Thérapeutiques (CPCET) and the biochemical laboratory of La Timone Hospital for blood sampling and analyses. The authors also thank the excellent cooperation of Dr. Thorsten Feiweier and Siemens AG (Erlangen, Germany), especially for the sequence used in DTI assessment (WIP 511c), the Assistance Publique des Hôpitaux de Marseille (APHM), and all the subjects who participated in the present study.

This study was supported by the Centre National de la Recherche Scientifique (CNRS UMR 7339) and Ajinomoto.

The authors' contributions are as follows: significant manuscript writer, A. F., D. B., J. G.; significant manuscript reviewer/reviser, A. F., G. D., J. W., J. P. M., H. B., A. L. T., D. B., J. G.; concept and design, A. F., J. P. M., H. B., D. B., J. G.; data acquisition, A. F., J. W.; data analysis and interpretation, A. F., G. D., A. L. T., D. B., J. G.; statistical expertise, A. F., J. G.

No conflicts of interest, financial or otherwise, are declared by the authors.

The results of the present study do not constitute endorsement by the American College of Sports Medicine.

## REFERENCES

1. Adams GR, Harris RT, Woodard D, Dudley GA. Mapping of electrical muscle stimulation using MRI. *J Appl Physiol* (1985). 1993;74(2):532-7.

2. Aldayel A, Jubeau M, McGuigan M, Nosaka K. Comparison between alternating and pulsed current electrical muscle stimulation for muscle and systemic acute responses. *J Appl Physiol* (1985). 2010;109(3):735-44.

3. Aldayel A, Jubeau M, McGuigan MR, Nosaka K. Less indication of muscle damage in the second than initial electrical muscle stimulation bout consisting of isometric contractions of the knee extensors. *Eur J Appl Physiol*. 2010;108(4):709-17.

4. Babault N, Cometti G, Bernardin M, Pousson M, Chatard JC. Effects of electromyostimulation training on muscle strength and power of elite rugby players. *J Strength Cond Res*. 2007;21(2):431-7.

5. Black CD, Elder CP, Gorgey A, Dudley GA. High specific torque is related to lengthening contraction-induced skeletal muscle injury. *J Appl Physiol* (1985). 2008;104(3):639-47.

6. Black CD, McCully KK. Force per active area and muscle injury during electrically stimulated contractions. *Med Sci Sports Exerc.* 2008;40(9):1596-604.

7. Black CD, McCully KK. Muscle injury after repeated bouts of voluntary and electrically stimulated exercise. *Med Sci Sports Exerc.* 2008;40(9):1605-15.

8. Bojsen-Moller J, Hansen P, Aagaard P, Svantesson U, Kjaer M, Magnusson SP. Differential displacement of the human soleus and medial gastrocnemius aponeuroses during isometric plantar flexor contractions in vivo. *J Appl Physiol* (1985). 2004;97(5):1908-14.

9. Cermak NM, Noseworthy MD, Bourgeois JM, Tarnopolsky MA, Gibala MJ. Diffusion tensor MRI to assess skeletal muscle disruption following eccentric exercise. *Muscle Nerve*. 2012;46(1):42-50.

10. Gibson JN, Smith K, Rennie MJ. Prevention of disuse muscle atrophy by means of electrical stimulation: maintenance of protein synthesis. *Lancet*. 1988;2(8614):767-70.

11. Guilhem G, Hug F, Couturier A, et al. Effects of air-pulsed cryotherapy on neuromuscular recovery subsequent to exercise-induced muscle damage. *Am J Sports Med*. 2013;41(8):1942-51.

12. HajGhanbari B, Hamarneh G, Changizi N, Ward AD, Reid WD. MRI-based 3D shape analysis of thigh muscles patients with chronic obstructive pulmonary disease versus healthy adults. *Acad Radiol*. 2011;18(2):155-66.

13. Heemskerk AM, Strijkers GJ, Drost MR, van Bochove GS, Nicolay K. Skeletal muscle degeneration and regeneration after femoral artery ligation in mice: monitoring with diffusion MR imaging. *Radiology*. 2007;243(2):413-21.

14. Hultman E, Sjöholm H, Jäderholm-Ek I, Krynicki J. Evaluation of methods for electrical stimulation of human skeletal muscle in situ. *Pflugers Arch*. 1983;398(2):139-41.

15. Jubeau M, Muthalib M, Millet GY, Maffiuletti NA, Nosaka K. Comparison in muscle damage between maximal voluntary and electrically evoked isometric contractions of the elbow flexors. *Eur J Appl Physiol*. 2012;112(2):429-38.

16. Jubeau M, Sartorio A, Marinone PG, et al. Comparison between voluntary and stimulated contractions of the quadriceps femoris for growth hormone response and muscle damage. *J Appl Physiol* (1985). 2008;104(1):75-81.

17. Kim HK, Laor T, Horn PS, Racadio JM, Wong B, Dardzinski BJ. T2 mapping in Duchenne muscular dystrophy: distribution of disease activity and correlation with clinical assessments. *Radiology*. 2010;255(3):899-908.

18. LeBlanc AD, Jaweed M, Evans H. Evaluation of muscle injury using magnetic resonance imaging. *Clin J Sport Med*. 1993;3(1):26-30.

19. Lieber RL, Fridén J. Muscle damage is not a function of muscle force but active muscle strain. *J Appl Physiol* (1985). 1993;74(2):520-6.

20. Longwei X. Clinical application of diffusion tensor magnetic resonance imaging in skeletal muscle. *Muscles Ligaments Tendons J*. 2012;2(1):19-24.

21. Maas H, Sandercock TG. Force transmission between synergistic skeletal muscles through connective tissue linkages. *J Biomed Biotechnol*. 2010;2010:575-672.

22. Mackey AL, Bojsen-Moller J, Qvortrup K, et al. Evidence of skeletal muscle damage following electrically stimulated isometric muscle contractions in humans. *J Appl Physiol* (1985). 2008;105(5):1620-7.

23. Mackey AL, Brandstetter S, Schjerling P, et al. Sequenced response of extracellular matrix deadhesion and fibrotic regulators after muscle damage is involved in protection against future injury in human skeletal muscle. *FASEB J*. 2011;25(6):1943-59.

24. Maffiuletti NA. Physiological and methodological considerations for the use of neuromuscular electrical stimulation. *Eur J Appl Physiol*. 2010;110(2):223-34.

25. Marqueste T, Giannesini B, Fur YL, Cozzone PJ, Bendahan D. Comparative MRI analysis of T2 changes associated with single and
repeated bouts of downhill running leading to eccentric-induced
muscle damage. *J Appl Physiol* (1985). 2008;105(1):299-307.

26. McMillan AB, Shi D, Pratt SJ, Lovering RM. Diffusion tensor MRI to assess damage in healthy and dystrophic skeletal muscle after lengthening contractions. *J Biomed Biotechnol*. 2011;2011:970726.

27. Morelli JN, Runge VM, Feiweier T, Kirsch JE, Williams KW, Attenberger UI. Evaluation of a modified Stejskal-Tanner diffusion encoding scheme, permitting a marked reduction in TE, in diffusion-weighted imaging of stroke patients at 3 T. *Invest Radiol. 2010;45(1):29-35.*

28. Nordez A, Jolivet E, Südhoff I, Bonneau D, de Guise JA, Skalli W. Comparison of methods to assess quadriceps muscle volume using magnetic resonance imaging. *J Magn Reson Imaging*. 2009;30(5):1116-23.

29. Nosaka K, Newton M, Sacco P. Delayed-onset muscle soreness does not reflect the magnitude of eccentric exercise-induced muscle damage. *Scand J Med Sci Sports*. 2002;12(6):337-46.

30. Prior BM, Jayaraman RC, Reid RW, et al. Biarticular and monoarticular muscle activation and injury in human quadriceps muscle. *Eur J Appl Physiol*. 2001;85(1-2):185-90.

31. Rodriguez-Falces J, Place N. Recruitment order of quadriceps motor units: femoral nerve vs. direct quadriceps stimulation. *Eur J Appl Physiol*. 2013;113(12):3069-77.

32. Speer KP, Lohnes J, Garrett WE Jr. Radiographic imaging of muscle strain injury. *Am J Sports Med*. 1993;21(1):89-95.

33. Stejskal EO, Tanner JE. Spin diffusion measurements: spin echoes in the presence of a time dependent field gradient. *J Chem Phys*. 1965;42:288-92.

34. Theurel J, Lepers R, Pardon L, Maffiuletti NA. Differences in cardiorespiratory and neuromuscular responses between voluntary and stimulated contractions of the quadriceps femoris muscle. *Respir Physiol Neurobiol*. 2007;157(2-3):341-7.

35. Vanderthommen M, Depresseux JC, Dauchat L, Degueldre C, Croisier JL, Crielaard JM. Spatial distribution of blood flow in electrically stimulated human muscle: a positron emission tomography study. *Muscle Nerve*. 2000;23(4):482-9.

36. Warren GL, Lowe DA, Armstrong RB. Measurement tools used in the study of eccentric contraction-induced injury. *Sports Med*. 1999;27(1):43-59.

37. Yanagisawa O, Kurihara T, Kobayashi N, Fukubayashi T. Strenuous resistance exercise effects on magnetic resonance diffusion parameters and muscle-tendon function in human skeletal muscle. *J Magn Reson Imaging*. 2011;34(4):887-94.

38. Zaraiskaya T, Kumbhare D, Noseworthy MD. Diffusion tensor imaging in evaluation of human skeletal muscle injury. *J Magn Reson Imaging*. 2006;24(2):402-8.

39. Zory R, Boërio D, Jubeau M, Maffiuletti NA. Central and peripheral fatigue of the knee extensor muscles induced by electromyostimulation. *Int J Sports Med*. 2005;26(10):847-53.