Participants.

The Institutional Review Board at Purdue University reviewed and approved all experimental procedures, and verbal and written informed consent were obtained from all participants. Eleven young male (n = 9) and female (n = 2) adults (means ± SE: age 23.8 ± 0.6 yr, body mass 72.4 ± 1.9 kg, height 175 ± 1.3 cm) volunteered to participate in this study. Participants were asked to fill out a medical history questionnaire. Individuals were excluded if they were obese (body mass index > 30 kg/m²), used tobacco products, were diabetic, were taking any medication other than birth control, participated in leg resistance exercise in the previous 6 mo, and performed endurance exercise for >3 days/week. Female participants were tested during the early follicular phase of their menstrual cycle (days 1–7) or during the placebo or no-pill week if they were taking oral contraceptives. All participants were asked to refrain from taking oral or topical analgesics, vitamins, and antioxidants for the duration of the study.

Experimental design.

Participants were asked to report to the laboratory on 10 different occasions over a 3- to 4-wk period. An overview of the experimental protocol is shown in Fig. 1. On the first experimental visit, a biopsy was obtained from the vastus lateralis muscle of one randomly selected thigh to serve as the resting baseline sample. The individuals were allowed to recover for at least 1 wk before the second experimental visit. The purpose of visits 2 and 3 was to familiarize the participants with the functional test on the isokinetic dynamometer as well as with the assessment of muscle soreness. These visits were separated by at least 24 h. On visit 4, participants underwent baseline assessment of muscle strength, fatigability, and soreness as described in detail below. At least 48 h after visit 4, participants reported back to the laboratory to perform a bout of eccentric exercise on the isokinetic dynamometer. A 355-kcal defined formula diet (Ensure; Abbott Laboratories) was given to participants the day before the session, and they were instructed to consume it at least 2 h before the onset of the experiment. Upon completion of the eccentric exercise bout, each thigh was assigned to receive either HT or a thermoneutral control intervention with a counterbalanced design. The heat and control treatments began ~10 min after the end of the exercise bout and lasted 90 min. The treatment sessions were repeated daily for 4 consecutive days. Muscle biopsies were taken from both thighs at day 1 (24 h) and day 5 (120 h) after the eccentric exercise bout. Muscle function and perceived soreness were reassessed throughout the recovery period as outlined in Fig. 1. Participants were instructed to fast for 10–11 h before undergoing muscle biopsies and to eat a light meal before the other experimental visits. Participants were also asked not to perform massage or foam rolling in the thighs and to refrain from local application of ice or heat throughout the duration of the study.

Eccentric exercise.

The exercise bout consisted of 300 maximal voluntary eccentric contractions at an angular velocity of 30°/s (20 sets, 15 repetitions/set) with the knee extensor muscles of each leg using an isokinetic dynamometer (Humac NORM; Computer Sports Medicine, Stoughton, MA). As demonstrated in detail by others (26, 38), this protocol has been shown to induce several manifestations of exercise-induced muscle damage, including impaired muscle function and a pronounced elevation in local pain. After warming up on an unloaded cycle ergometer for 5 min, subjects were positioned on the chair of the isokinetic dynamometer and initially performed 75 eccentric knee extensions (5 sets of 15 repetitions with 30 s rest between sets) with one randomly selected leg. After 2–3 min of rest, subjects followed the same protocol with the other leg and the procedure was repeated four times. Participants were asked to resist as the lever pulled their partially extended leg from 35° of knee flexion (0° = full extension) to 105° of knee flexion, resulting in a range of motion of 70°. Consistent verbal encouragement was provided throughout the exercise bout.

Heat treatment.

Local heating and the thermoneutral intervention were applied with a water-circulating garment customized to cover the thighs and buttocks (Med-Eng, Ottawa, ON, Canada) as described previously (20). The garment consists of a network of medical-grade polyvinyl chloride tubing sewn onto tight-fitting elastic fabric and connected to a water circulator. In the thigh assigned to receive HT, water at 54–55°C was circulated through the garment for 90 min to increase skin temperature to ~39.5–40°C (20). In the opposite thigh, thermoneutral water (32–33°C) was circulated to clamp skin temperature at ~33°C for the entire duration of the protocol. The length of the treatment sessions was based on our recent report that exposure to local HT for 90 min enhances the expression of angiogenic regulators and stress management genes in human skeletal muscle (20). Skin thermocouples were taped to each thigh and allowed skin temperature to be monitored continuously during the treatment sessions.

Muscle biopsies.

Muscle biopsies (~200 mg) were taken from the vastus lateralis muscle after 30 min of rest in the supine position as described previously (20). Under local anesthesia (lidocaine hydrochloride; Hospira, Lake Forest, IL), a small incision was made in the skin and fascia, followed by the insertion of a 5-mm Bergström biopsy needle (Pelomi Medical, Albruslund, Denmark). The biopsy specimens were promptly weighed, cleared of visible fat and connective tissue, and divided into four sections. The portion designated for cryosectioning was placed in a disposable base mold, covered with Tissue-Tek optimal cutting temperature compound and frozen in liquid nitrogen-cooled isopentane (49). The other sections were immediately frozen in liquid nitrogen and stored at −80°C until RNA extraction or Western blot analysis. Biopsies were acquired from separate incisions, ~2–3 cm apart. Leg order for biopsy sampling was randomized for each time point. All muscle biopsies were performed between 7:30 AM and 9:00 AM.

Assessment of muscle strength and fatigability.

Maximal knee extensor strength and fatigue resistance were assessed using an isokinetic dynamometer. Participants were familiarized with the testing procedures twice before the baseline assessment. Each session was preceded by a standardized protocol consisting of 5 min of pedaling on a cycle ergometer. After the warm-up, participants were positioned on the chair of the isokinetic dynamometer and straps were fastened at the waist and shoulders and across the thigh to maintain a stable body position. The dynamometer settings were recorded on the first familiarization session and replicated in subsequent tests. The familiarization protocol included a set of 5–10 concentric knee extensions at 60–70% of the estimated maximal effort at an angular velocity of 180°/s, a set of three maximal contractions at an angular velocity of 180°/s, a set of three maximal contractions at an angular velocity of 60°/s, and a final set of 28 consecutive maximal contractions at 180°/s.

Maximal isokinetic strength of the knee extensors was assessed at baseline (visit 4) and daily throughout the recovery period. Fatigue resistance was assessed at baseline and 1 and 4 days after the eccentric exercise bout. Testing was performed on both legs, with the order of the testing counterbalanced. In each experimental session, participants were allowed to warm up for 5 min on a cycle ergometer and were then positioned on the chair of the isokinetic dynamometer with the identical apparatus setting predetermined at the first familiarization visit. Maximal isokinetic strength was assessed at two angular velocities: 180°/s and 60°/s. Participants were asked to complete three maximal consecutive contractions at each angular velocity, with a resting period of 30 s between velocities and 3 min between limbs. Peak isokinetic torque was defined as the average of the two highest attained values. Once both limbs had been tested for maximal strength, participants were allowed to rest for ~3 min and were then asked to perform a bout consisting of 28 consecutive maximal contractions at 180°/s. A resting period of 10 min was allowed between limbs. The total work performed during the bout was computed and used as a measure of fatigue resistance of the knee extensors. The reliability of muscle performance assessment was determined by comparing the results of the second familiarization visit (visit 3) and the baseline testing visit (visit 4). Both measures of muscle strength and fatigue resistance were found to be highly reproducible [retest correlations: 1) peak torque at 180°/s: 0.91, 2) peak torque at 60°/s: 0.92, 3) total work completed during the fatigue bout: 0.97].

Perceived muscle soreness.

Participants were asked to evaluate knee extensor muscle soreness on a 10-cm visual analog scale (0 = no soreness, 10 = extreme soreness) after stepping on and off a 40-cm (women) or 45-cm (men) chair three times.

RNA extraction.

Total RNA from the skeletal muscle biopsies was prepared with the TRIzol Reagent (Invitrogen, Life Technologies, Carlsbad, CA) and quantified spectrophotometrically (Nanodrop 3000; ThermoFisher), as described previously (20). The quality and integrity (RNA integrity number of 8.2 ± 0.1) of extracted RNA (276.9 ± 26.4 ng/μl) were evaluated with an RNA 6000 Nano LabChip kit on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). cDNA was prepared with the RT² First Strand Kit following the manufacturer’s instructions (Qiagen, Valencia, CA).

Skeletal muscle gene expression.

The expression of nine select genes related to angiogenesis, myogenesis, inflammation, and stress management was determined with a custom RT² Profiler PCR array kit (Qiagen) and the Roche LightCycler 480 PCR System (Roche Diagnostics, Indianapolis, IN). Duplicate samples from each subject (baseline and day 1 and day 5 after eccentric exercise) were loaded in the same array plate. The list of genes analyzed is shown in Table 1 and included the housekeeping gene 18S ribosomal RNA (18S rRNA) as well as a reverse transcription control, a positive PCR control, and a genomic DNA control. Data were analyzed with the GeneGlobe Data Analysis Center (Qiagen). 18S rRNA was used as the internal reference gene and proved stable across all time points. The comparative C_t method (where C_t is threshold cycle) was used to calculate the changes in gene expression of each target mRNA relative to the baseline sample (22).

Protein extraction.

Frozen muscle samples (~30 mg) were homogenized in ice-cold homogenization buffer containing 0.5 M Tris·HCl, pH 7.4, 1.5 M NaCl, 2.5% deoxycholic acid, 10% NP-40, and 10 mM EDTA (RIPA Lysis Buffer; EMD Milipore) with freshly added protease inhibitor cocktail (Sigma-Aldrich catalog no. P8340) at a 1:15 dilution of wet muscle weight with a bead mill homogenizer (Bead Ruptor 12; Omni International). The resulting homogenate was clarified by centrifugation (13,500 g) for 20 min at 4°C. The supernatant was collected, and the protein concentration of each sample (~5 µg/µl) was determined with a BCA protein assay kit (Thermo Scientific). All samples were subsequently diluted with 1× PBS and subsequently mixed with either reducing sample buffer (4× Laemmli sample buffer with 10% 2-mercaptoethanol) or nonreducing sample buffer (4× Laemmli sample buffer). Afterwards, samples were heated for 5 min at 95°C, divided into small aliquots, and stored at −80°C.

Western blot analysis.

Twenty-five micrograms of proteins was separated by SDS-PAGE on precast Stain-Free 4–15% gels (Bio-Rad) and transferred to polyvinylidene fluoride membranes with the Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were subsequently blocked with 5% nonfat milk in 1× Tris-buffered saline-Tween 20 (TBST; 1% Tween 20) solution for 1 h at room temperature and incubated overnight at 4°C with primary antibodies diluted in blocking buffer. Details of the primary antibodies and recombinant proteins are provided in Table 2. The membranes were washed with 1× TBST at room temperature for 3 × 10 min, incubated with horseradish peroxidase-conjugated secondary antibodies diluted in 1× TBST for 1 h at room temperature, and then washed with 1× TBST at least 3 × 10 min before being exposed to an enhanced chemiluminescence solution (Clarity Western ECL; Bio-Rad) for 5 min. Membranes were visualized with a densitometer (ChemiDoc Touch Imaging System; Bio-Rad), and band densities were determined with image analysis software (Image Laboratory V5.0; Bio-Rad). PageRuler Prestained Protein Ladder (Thermo Fisher) was used as a molecular weight marker. Control for equal loading was performed with stain-free technology, and total protein normalization was used to calculate changes in the expression of each target protein relative to the baseline sample (12).

Immunohistochemistry.

Transverse serial sections (10 µm) of muscle biopsy samples were cut with a Leica CM1850 cryostat (Leica, Wetzlar, Germany) at −25°C, mounted on frosted microscope slides (Thermo Scientific), air-dried for 0.5–1 h at room temperature, and stored at −80°C for subsequent analyses. Frozen sections were briefly exposed to room air and fixed with 4% paraformaldehyde for 5 min. After 2 × 3 min washes with 1× PBS, the slides were incubated with blocking buffer (5% goat serum, 2% bovine serum albumin, 0.1% Triton X-100, and 0.1% azide in PBS) for 1 h at room temperature (52). Thereafter, sections were incubated with primary antibodies diluted in blocking buffer for 3 h at room temperature. Macrophages were stained with an antibody for CD68 (mouse IgG2b, 1:100; R&D Systems catalog no. MAB20401), whereas laminin (rabbit IgG1, 1:500; Abcam catalog no. ab11575) was added for distinction of the myofiber sarcolemma. Identification of fiber type-specific capillaries was performed with antibodies against CD31 (mouse IgG1, 1:100; BD Biosciences catalog no. 550300), dystrophin (rabbit IgG1, 1:100; Abcam catalog no. ab15277), and myosin heavy chain type I (MHC I) (mouse IgG2b, supernatant 1:100; Developmental Studies Hybridoma Bank catalog no. BA-D5). After 2 × 5 min washes with 1× PBS, sections were stained with appropriate secondary antibodies [Alexa 350 goat anti-rabbit IgG (1:500, catalog no. A-11069), Alexa 488 goat anti-rabbit IgG (1:1,000, A-11008), Alexa 488 goat anti-mouse IgG2b (1:1,000, A-21141), and Alexa 568 goat anti-mouse IgG1 (1:1,000, A-21124); Thermo Fisher Scientific], diluted in 1× PBS for 1 h at room temperature. Nuclei were labeled with 4′,6-diamidino-2-phenylindole. After 4 × 5 min washes, slides were briefly dried and mounted with fluorescent mounting medium (Dako) and the edges were sealed with nail polish (Sally Hansen Hard as Nails). Negative controls for the primary antibodies against CD31 and CD68 were used to ensure specificity of staining. Slides were viewed at ×20 magnification with an Olympus BX53 fluorescence microscope equipped with an Olympus DP72 digital camera and cellSens Dimension software. The entire specimen cross section was initially selected with the stage navigator. The multichannel image was then acquired, and two images from each channel were merged with ImageJ software (National Institutes of Health). Histological analysis was not performed in 3 of 55 samples because of insufficient muscle yield.

Analysis of immunofluorescence images.

Analyses of immunofluorescence images were carried out with Adobe Photoshop CC 2015. For the quantification of fiber type-specific capillarization, all internal fibers (not bordering on a fascicle) in a cross section were initially counted (an average of 173 ± 95 fibers for type I and 217 ± 121 for type II muscle fibers). A total of 50 type I and 50 type II muscle fibers were then randomly selected for analysis (37). All capillaries within a distance of 5 µm from each fiber were counted for the determination of the number of capillaries around each fiber (CAF) (55). To evaluate the distribution and perform the quantification of macrophages, an average of 736 ± 297 fibers were analyzed. Macrophages were identified when the fluorescent signal for CD68 clearly surrounded (11) or entirely covered a nucleus. All immunofluorescent images were masked for both group and time point before analysis.

Statistical analysis.

All statistical analyses were conducted with SAS (version 9.4; SAS Institute), with results expressed as means ± SE. The Kolmogorov-Smirnov test was used to assess the distribution of the data. Data exhibiting skewed distribution were log-transformed before statistical analysis. A two-way repeated-measures ANOVA was employed to compare the changes in skeletal muscle performance and soreness, gene and protein expression responses, and changes in the content of CD68⁺ immunoreactive macrophages and CAF between the leg exposed to HT and the control leg. Post hoc analysis (Tukey) was performed when appropriate. For all analyses, P < 0.05 was considered statistically significant.

Performance during eccentric exercise.

Figure 2 depicts the amount of work completed during each of the 20 sets of eccentric contractions by the thigh that was later assigned to receive HT and by the thigh that was subsequently allocated to the thermoneutral control treatment. On average, the thigh randomized to receive HT performed slightly more work (4.6%) than the opposite thigh (control 1,649 ± 108 J vs. HT 1,724 ± 115 J). All participants were right-leg dominant (based on kicking preference). Six participants were assigned to receive HT on their dominant leg, and five individuals had their dominant leg treated with the control regimen.

Changes in muscle function in response to muscle damage and recovery.

The responses of isokinetic peak torque, total work completed during the fatigue bout, and perceived muscle soreness are displayed in Figs. 3–5. There were no significant differences between thighs for baseline measures of isokinetic peak torque at 180°/s (control 139 ± 2.3 Nm vs. HT 143 ± 1.9 Nm, P = 0.29) or 60°/s (control 181 ± 3.2 Nm vs. HT 181 ± 2.4 Nm, P = 0.37) and total work amount completed during the fatigue trial (control 3,531 ± 56 J vs. HT 3,575 ± 60 J, P = 0.60). Peak torque decreased, on average, by ~45% in the first 2 days after the eccentric exercise protocol and began to recover progressively starting at day 3 (Fig. 3). There were no differences between thighs in the overall recovery profile of peak torque. Fatigue resistance, as determined by the total work amount completed during 28 consecutive maximal contractions at 180°/s, decreased by ~20% at day 1 after the completion of the eccentric exercise bout and remained below baseline values at day 4 (Fig. 4). The thigh exposed to HT had significantly greater fatigue resistance (i.e., lower reduction in total work relative to baseline) compared with the control thigh (P = 0.02, main effect). Perceived muscle soreness rose sharply after the eccentric exercise bout, reaching peak values at day 2 (Fig. 5). Exposure to HT tended to decrease the magnitude of muscle soreness relative to the control treatment (P = 0.053).

Gene expression.

Fold changes in gene expression from baseline for select angiogenic, myogenic, inflammatory, and stress management genes are shown in Fig. 6. At day 1, the mRNA expression of VEGF (control 0.79 ± 0.10 vs. HT 1.24 ± 0.12, P < 0.05) was significantly higher in the thigh exposed to HT compared with the control thigh. Conversely, at day 5, the mRNA expression of ANGPT1 (control 1.02 ± 0.13 vs. HT 0.78 ± 0.12, P < 0.05) was significantly lower in the thigh exposed to HT. The mRNA expression of CCL2 also was significantly lower in the thigh exposed to HT (P = 0.01, main effect).

Protein expression.

Figure 7 displays the fold changes in the protein levels of select angiogenic, myogenic, inflammatory, and stress management factors relative to the baseline sample. A significant increase in the expression of chemokine CX3CL1 was observed in the thigh exposed to HT compared with the control thigh on day 1 (control 0.87 ± 0.07 vs. HT 1.21 ± 0.13, P < 0.05). The expression of VEGF (P = 0.005, main effect), ANGPT1 (P = 0.045, main effect), and CCL2 (P = 0.02, main effect) was higher in the thigh treated with HT compared with the control thigh.

Macrophage content.

Skeletal muscle cross sections were analyzed for the presence of CD68⁺ macrophages by immunohistochemistry (Fig. 8). The majority of CD68⁺ immunoreactive macrophages were detected between fibers (Fig. 8A). Intracellular staining for CD68 was only detected in a few fibers. An increasing trend in the number of CD68⁺ cells per 100 muscle fibers was noted after exposure to eccentric exercise (P = 0.071; Fig. 8B). On average, macrophage content increased by 44% on day 1 and by 130% on day 5. There was no treatment effect or treatment × time interaction for this variable.

Skeletal muscle capillarization.

One representative image of a muscle cross section stained for MHC I, CD31, and dystrophin is displayed in Fig. 9A. There was no treatment effect or treatment × time interaction for CAF. A significant decrease in CAF was observed at day 5 after the eccentric exercise bout in type II fibers (P < 0.05).

Limitations.

One particular challenge in the design of studies aimed at assessing the impact of HT on recovery after exercise is that it is not possible to blind subjects to the exposure to heat. Although participants in the present study were not informed of the study hypothesis and a control intervention was used for comparison, we did not examine the potential influence of the placebo effect on the observed responses. Accumulating evidence indicates that therapeutic effects of popular recovery modalities, such as cryotherapy, stem at least partially from a placebo effect (53, 54). These important observations emphasize the need for future studies on the effects of HT to implement an effective placebo-controlled design.

Recovery from intense eccentric exercise entails a complex, well-orchestrated response that may persist for several weeks. Insights into the mechanisms underlying the beneficial effects of HT were limited in the present study because muscle biopsies were harvested only in the immediate (day 1) and subacute (day 5) phases of recovery. Indeed, some of the manifestations of exercise-induced muscle damage, including the decline in force and power output, increased soreness, and reduced capillarization, were not fully resolved by day 5. Future studies should examine the impact of repeated local HT on the later phases of recovery.

Perspectives and significance.

Our results show that exposure to HT immediately after and for 4 consecutive days after a maximal bout of eccentric exercise in humans hastens recovery of fatigue resistance and tends to reduce perceived soreness. These findings are in line with a growing body of literature supporting a beneficial impact of HT on recovery after noninjurious exercise (8) as well as severe muscle injuries (19, 32, 33, 44, 48). Additional studies are warranted to determine whether repeated exposure to local HT amplifies the skeletal muscle adaptations to exercise training in humans.