Effects of essential amino acid supplementation on musculotendinous recovery following eccentric plantar flexor exercise: a randomized controlled trial

Abstract

Background

Exercise-induced muscle damage (EIMD) resulting from eccentric contractions leads to transient impairments in muscle function. Essential amino acids (EAAs) stimulate muscle protein synthesis and may support recovery following damaging exercise. However, limited research has examined the effects of EAAs on muscle function following eccentric plantar flexor exercise. The purpose of this study was to examine the effects of EAA supplementation on indirect markers of muscle and musculotendinous recovery following EIMD.

Methods

Thirty-six recreationally active males (age: 21.2 ± 2.7 years) were randomly assigned to an EAA group (10 g of EAAs), placebo (10 g of maltodextrin), or control (no supplementation). Supplements were consumed 30 minutes before and immediately after an eccentric plantar flexor protocol (4 × 50 repetitions followed by one set to failure) and during the 72 hours recovery period. Indirect markers of muscle damage were assessed pre-exercise, immediately post-exercise, and 24, 48, and 72 hours post-exercise, consisting of perceived soreness (NPRS), pain pressure threshold (PPT), calf muscle thickness and Achilles tendon thickness (ultrasound), and calf circumference.

Results

The eccentric protocol elicited responses consistent with EIMD, including increased soreness (p < 0.001), increased calf muscle thickness and circumference (p < 0.001), and reduced PPT (p = 0.018). EAA supplementation attenuated soreness at 24 hours compared with placebo. Calf muscle thickness increased following exercise in both groups; however, swelling returned to baseline by 72 hours in the EAA group but remained elevated in placebo (Condition × Time, p < 0.001). Achilles tendon thickness decreased immediately post-exercise (p = 0.005) but was not influenced by supplementation.

Conclusions

EAA supplementation modestly reduced soreness and was associated with faster recovery of muscle swelling but did not influence tendon morphology within 72 hours.

1. Introduction

Performing repeated high-force eccentric contractions lead to structural disruption of skeletal muscle fibers, resulting in exercise-induced muscle damage (EIMD) [1]. EIMD initiates a cascade of events characterized by sarcomere disruption, calcium dysregulation, inflammatory cell infiltration, and activation of proteolytic pathways [1,2]. Although muscle protein synthesis (MPS) is elevated following damaging exercise, the early recovery phase is characterized by ongoing sarcomeric disruption, excitation‒contraction uncoupling, and inflammatory signaling that collectively impair contractile function before structural repair is complete [1–3]. Eccentric contractions, in particular, induce non-uniform sarcomere lengthening, Z-line streaming, and cytoskeletal perturbations that compromise force transmission within the musculotendinous unit [2]. Consequently, reductions in maximal strength, increased muscle soreness, swelling, elevated biomarkers of muscle damage, and impairments in functional performance such as jump height and rate of force development are commonly observed [3–5]. For example, eccentric loading of the plantar flexors has been shown to significantly elevate creatine kinase, increase perceived soreness, and reduce range of motion and force output within 24–72 hours post-exercise [6,7]. Therefore, nutritional strategies that increase essential amino acids (EAA) availability during this critical window of remodeling may enhance myofibrillar repair processes and facilitate a rapid restoration of muscle function following EIMD.

Dietary protein supplementation has consistently been shown to augment MPS by increasing circulating amino acid concentrations and activating anabolic signaling pathways, particularly the mechanistic target of rapamycin complex 1 (mTORC1) [7–9]. The nine EAAs, leucine, isoleucine, valine, lysine, methionine, threonine, phenylalanine, tryptophan, and histidine, are required substrates for new muscle protein formation [10]. Importantly, leucine acts as a key regulator of mTORC1 signaling, serving as a molecular trigger for translation initiation and ribosomal activation [11,12]. While whole protein sources contain EAAs, supplementation with free-form EAAs may provide a greater rise in plasma amino acid concentrations compared to when the same EAAs are ingested as a part of whole protein [13,14]. This distinction reflects differences in absorption kinetics rather than demonstrated superiority in recovery outcomes, and the present study was not designed to directly compare EAA supplementation with a whole-protein source.

Previous investigations have demonstrated that ingestion of EAAs stimulates MPS both at rest and following resistance exercise [15]. In contrast to intact proteins, free-form EAAs are rapidly absorbed and do not require digestion, leading to a more immediate elevation in plasma amino acid availability [10,15]. Lees et al. [16] reported that a leucine-enriched EAA gel significantly elevated plasma leucine concentrations and augmented mTOR pathway activation 2 hours post-ingestion, irrespective of age, supporting the capacity of targeted EAA supplementation to stimulate anabolic signaling following resistance exercise. Similarly, studies examining post-exercise recovery suggest that EAA supplementation may attenuate markers of muscle damage and improve recovery of strength and functional performance [17,18]. The proposed mechanisms include enhanced myofibrillar protein synthesis, modulation of inflammatory responses, and potential reduction in structural damage [19–21].

While previous research has examined the effects of whey protein and other intact protein sources on recovery from eccentric exercise [22,23], fewer studies have isolated the effects of EAAs alone in the context of acute musculotendinous recovery, particularly in the gastrocnemius musculature. Of particular interest is the triceps surae complex as it plays a critical role in locomotion, force transmission, and elastic energy storage during activities such as walking and running [24]. Given its role in coordinated muscle‒tendon function and force production, determining whether EAA supplementation improves recovery of tissue mechanics, and pain may help clarify the role of amino acid availability in restoring function after exercise-induced muscle damage.

Therefore, the purpose of this study was to identify and compare the effects of EAA supplementation versus placebo on indirect markers of musculotendinous unit recovery following eccentric plantar flexor exercise. It was hypothesized that EAA supplementation would attenuate perceived muscle soreness and improve mechanical properties of the musculotendinous unit compared to placebo. Because EAA availability is thought to influence early-phase muscle protein turnover and modulation of inflammatory signaling, it was expected that these effects would be reflected in the specific indirect markers assessed in this study, including perceived soreness (NPRS), pain pressure threshold, calf muscle thickness, Achilles tendon thickness, and calf circumference, rather than in a direct measure of muscle damage itself.

2. Materials and methods

2.1. Study design

Participants reported to the Human Performance Lab for Visit 1, where they reviewed and signed an informed consent document prior to participation. Eligibility was determined using the Health History Questionnaire (HHQ) and the Physical Activity Readiness Questionnaire (PAR-Q+). Participants also completed a dietary recall. Eligible individuals were scheduled for four subsequent visits and randomly assigned to one of three groups: EAA supplementation, placebo, or control. Participants were instructed to refrain from structured exercise and the use of non-steroidal anti-inflammatory drugs between Visits 1 and 5 to minimize confounding effects on outcome measures. If soreness became intolerable, participants were permitted to use over-the-counter non-steroidal anti-inflammatory medications or acetaminophen according to manufacturer recommendations and were required to document all usage and report it to the research team. The participants voluntarily signed an informed consent form, and the procedures were approved by the university Institutional Review Board (ID: 51453564).

At Visit 2, participants in the EAA and placebo groups consumed two 10-g servings of their assigned supplement. The EAA group received 10 g of EAAs (provided by Optimal Labs, LLC with leucine content of 19.64%), whereas the placebo group received 10 g of taste-matched placebo (maltodextrin) containing no amino acids. Supplements were mixed in 300 mL of water. One serving was ingested 30 minutes prior to testing and the second immediately following the testing session. The control group received no supplementation. Prior to supplementation, anthropometric assessments were obtained. Height and body mass were measured using a stadiometer and calibrated scale (Detecto Physician Scale, Detecto, Webb City, MO, USA).

Participants then completed a standardized testing battery consisting of perceived muscle soreness, pain pressure threshold assessment at the gastrocnemius, calf circumference, muscle thickness, Achilles tendon thickness, and the Numeric Pain Rating Scale (NPRS). Following baseline assessments, participants in the EAA and placebo groups completed an eccentric plantar flexor exercise protocol.

The EIMD protocol targeted the dominant leg and consisted of 4 sets of 50 repetitions with last fifth set completed to failure, of assisted isotonic plantar flexion concentric and eccentric muscle actions with a weight vest on equivalent to approximately 40% of each participants body mass. Each repetition consisted of a one-second concentric contraction to raise the heel followed by a three-second eccentric lowering phase until the heel descended below neutral (0°). Participants used an overhead pull-up bar to assist with returning to the starting position, thereby minimizing concentric load. This protocol was adapted from a previously used eccentric plantar flexor exercise procedure shown to elicit responses consistent with EIMD in a similar recreationally active population (Vasenina et al., 2022); as with that protocol, the present study relies on indirect markers rather than direct structural or biochemical confirmation of muscle damage. Immediately following the protocol, the testing battery was repeated and post-exercise supplementation was administered when applicable.

Participants returned to the laboratory 24, 48, and 72 hours post-exercise (Visits 3–5) to repeat the standardized testing battery and supplementation procedures. At Visit 5, participants completed three-day dietary logs.

2.2. Procedures

An a priori power analysis was performed using G*Power (Faul et al., 2007) to determine the minimum sample size necessary. Assuming a moderate Cohen's f effect size of 0.25 with a type I error rate of 5% and a type II error rate of 20% (i.e. 1-β [0.80]) for a two-way (3 × 5) repeated measures mixed factorial ANOVA (i.e. within-between interaction), a total sample size of 27 was needed to achieve statistical power (i.e. 80%). Thus, a convenience sample of 44 males between the ages of 18–35 years old was recruited from the University of Tampa and surrounding areas for participation in this study of musculotendinous recovery following eccentric exercise. Of the 44 participants enrolled, 36 completed the study [age: 21.2 (±2.7) years, height: 178.4 (±6.1) cm, body mass: 78.0 (±11.3) kg; seven participants withdrew due to scheduling conflicts that prevented attendance at follow-up visits, and one participant withdrew due to increased perceived lower extremity soreness following the exercise protocol, without evidence of musculoskeletal injury or adverse clinical symptoms. No significant between-group differences were found for age, height, or body mass (p > 0.05). All participants were recreationally active (defined as engaging in exercise at least 3 times per week), free of any physical limitations, and answered “no” to all questions on the PAR-Q+.

2.3. Supplementation

Thirty minutes before EIMD and immediately after, participants consumed 10 g of EAAs (Optimal Labs, LLC with leucine content of 19.64%) in the EAA group, and 10 g of taste-matched placebo (maltodextrin) containing no amino acids in the placebo group. Supplements were mixed in 300 mL of water. To maintain double-blinded procedures, supplements were pre-packaged and coded by a researcher not involved in data collection or analyzes, and both participants and investigators remained blinded to group allocation throughout the study. For the two days following EIMD, the doses were consumed 30 minutes prior to lab visits and immediately after the lab visit. The remaining supplement was consumed prior to final measurements at 72 hours post-exercise. This is based on recent work demonstrating the effect of EAA consumption on MPS following EIMD [17,25].

2.4. Measures

2.4.1. Anthropometrics

Height and weight were assessed using a stadiometer and scale (Detecto Physician Scale, Detecto, Webb City, MO, USA). Participants were asked to remove shoes and hats for this assessment.

2.4.2. Numerical pain scale

Participants were instructed prior to Visit 2 on how to report pain using the Numeric Pain Rating Scale (NPRS) [26]. A standardized script was used to ensure consistency in instructions across participants. Individuals were asked to rate their least pain over the previous 24 hours, their worst pain over the previous 24 hours, and their current pain level using a 100-point scale, where 0 indicated no pain and 100 represented the most intense pain imaginable. Ratings for current, least, and worst pain were averaged to generate a composite pain score for statistical analyzes.

2.4.3. Pain pressure threshold

Pain pressure threshold was assessed using a digital pressure algometer (FPX 25, Wagner Instruments, Greenwich, CT, USA) fitted with a 1-cm diameter rubber probe. The device was applied perpendicular (90°) to the skin surface at the midpoint of the gastrocnemius muscle, corresponding to 70% of the distance from the medial tibiofemoral joint line to the center of the medial malleolus. Pressure was increased at a consistent rate until participants verbally indicated the first transition from pressure to pain by stating “stop” or “pain,” at which point the force application was immediately terminated. To minimize risk of tissue injury, a maximum cut-off value of 50 kg was established. Two trials were performed at the marked site in randomized order, separated by a 30-second rest interval. The mean of the two measurements was calculated and recorded in pounds for statistical analyzes. Although multiple investigators were involved in data collection across study procedures, all pain pressure threshold assessments were performed by the same investigator. Prior reliability testing conducted in our laboratory demonstrated excellent test–retest reliability for pain pressure threshold (ICC_2,k = 0.91).

2.4.4. Muscle thickness and calf circumference

Muscle thickness of the randomized limb was assessed using portable B-mode ultrasound (HS40; Samsung Healthcare, Ridgefield Park, NJ) equipped with a linear-array transducer (3–16 MHz). The measurement site was identified at 70% of the distance between the medial tibiofemoral joint line and the center of the medial malleolus. Ultrasound gel was applied to the marked location prior to image acquisition. Two images were obtained at each time point, and both the highest and mean values were recorded for analyzes. Prior reliability testing conducted in our laboratory demonstrated excellent test–retest reliability for ultrasound-derived muscle thickness (ICC_2,k ≥ 0.98).

Calf circumference was measured at the same anatomical location (70% of the distance between the medial tibiofemoral joint line and the medial malleolus) using a flexible tape measure (Gulick II Tape Measure; Country Technology Inc., Gays Mills, WI, USA). Two to three measurements were obtained per time point, and both the highest and mean values were recorded. Laboratory reliability testing indicated excellent test–retest reliability for calf circumference (ICC_2,k = 0.97).

2.4.5. Tendon thickness

Sagittal images of the Achilles tendon were collected, with the ankle passively dorsiflexed to approximately 90 degrees. Tendon thickness was measured at a reference point of 2 cm proximal to the superior aspect of the calcaneus [27]. The Achilles tendon was scanned using a B-mode US imaging device (HS40; Samsung Healthcare, Ridgefield Park, NJ) equipped with a linear-array transducer (3–16 MHz). All assessments were performed by the same investigator to minimize inter-rater variability. Prior reliability testing conducted in our laboratory demonstrated excellent test–retest reliability for Achilles tendon thickness (ICC_2,k = 0.99).

2.4.6. Dietary analysis

Participants were asked to record all food and beverage intake for three consecutive days using the MyFitnessPal dietary tracking application (MyFitnessPal Inc., San Francisco, CA, USA). Participants were instructed to log the type of food consumed, portion sizes, and preparation methods to improve the accuracy of the dietary records. Dietary data were exported from MyFitnessPal and analyzed to determine habitual nutrient intake. Variables extracted included total energy intake, macronutrient intake (protein, carbohydrates, and fat), macronutrient-derived energy (kcal), fiber, and selected micronutrients including vitamins A and C, calcium, iron, potassium, and sodium.

2.5. Statistical analyzes

2.5.1. Frequentist

Assumptions of normality (i.e. examination of skewness, kurtosis, and Shapiro‒Wilk tests of the residuals) and homogeneity of variance (Levene's test) were performed prior to performing the primary analyzes. To determine if there were any significant differences at baseline, separate one-way, 3 (Condition [EAA, Placebo, Control]), ANOVA models were performed for the descriptive characteristics and the nutritional content from the dietary logs. Separate two-way, 3 (Condition [EAA, Placebo, Control]) × 5 (Time [Pretest, Posttest, 24, 48, and 72 hours]), mixed factorial repeated-measures ANOVA models were used to examine potential mean differences for each dependent variable. Greenhouse-Geisser corrections were applied when Mauchly's test of sphericity was significant (Greenhouse & Geisser, 1959) [28]. Each significant ANOVA model was examined with follow-up, Bonferroni-adjusted one-way ANOVAs and/or repeated measures ANOVAs and are reported as the type I error rate (p value), 95% confidence interval (95% CI), and effect size (Hedges g). Partial eta-squared effect sizes ( ${\eta }_p^2$ ) were calculated for each ANOVA model where 0.01–0.06 was considered small, 0.06–0.14 was considered medium, and >0.14 was considered large. For each paired samples or independent samples t-tests, Hedge's g effect sizes were calculated where 0–0.2 was considered minimal, 0.2–0.5 was considered small, 0.5–0.8 was considered medium, >0.8 was considered large. All frequentist statistics were performed with IBM SPSS v. 30 (Armonk, NY) with a type I error rate of 0.05 for all significant comparisons.

2.5.2. Bayesian

For each significant ANOVA model, Bayesian paired samples and/or independent samples t-tests using Markov Chain Monte Carlo (MCMC) estimation with 100,000 iterations and default, weakly informative priors (prior = NULL) were also performed. These Bayesian analyzes were conducted as supplementary analyzes to complement the primary frequentist inferential framework by quantifying the posterior probability of the observed effect (i.e. the proportion of posterior estimates greater than zero) for significant pairwise comparisons identified from the ANOVA models. Thus, the Bayesian analyzes were not intended to replace the primary repeated-measures ANOVA approach or evaluate the full factorial model but rather to provide additional probabilistic information regarding statistically significant effects. For each Bayesian model, the burn-in was set to 1,000 iterations to help achieve a stationary distribution and reduce any potential bias of the MCMC estimates. Also, each model had three independent chains (i.e. 100,000 iterations saved per chain) and a thinning interval of one. Additionally, for variables with zero variance (e.g. certain NPRS time points), a small amount of Gaussian noise (SD = 0.05) was added prior to Bayesian estimation to permit MCMC estimation. To assess sampling quality (effective sample size [ESS] ≥ 400) [29] Markov chain convergence (scale reduction factor [R̂] = 1.00), and model fit (examination of the empirical cumulative distribution function [ECDF] plots), posterior predictive checks were visually assessed for each model. All Bayesian paired samples and/or independent samples t-test were reported as the probability of the effect occurring (Prob; 0%–100%). For each Bayesian paired samples and/or independent samples t-test, the Bayesian Estimation Supersedes the t-test (BEST) package was used and employs a Gibbs sampler [30]. All Bayesian statistics were performed in R studio v. 4.5.1 and each figure was constructed in GraphPad Prism 10.6.1 (Boston, MA). See supplementary material for the R-code used in this investigation.

3. Results

3.1. Baseline characteristics and posterior predictive checks

Table 1 includes the findings from the one-way ANOVA models for the baseline differences in descriptive characteristics and nutritional content from the dietary logs. There were significant (p = 0.024–0.037; ${\eta }_p^2$  = 0.186–0.207) differences between groups for carbohydrates (i.e. both grams and kcal). There were, however, no significant (p > 0.05) differences for the remaining variables. Throughout the supplemental Bayesian analyzes, there was adequate sampling quality for all Bayesian models, with effective sample sizes ≥ 400 and successful chain convergence (R̂ = 1.00), suggesting sufficient exploration of the posterior distributions and reliable variance estimation.

Table 1.

Displays the findings from the one-way analysis of variance (ANOVA) models that were performed for the descriptive characteristics and the nutritional content from the dietary logs. Specifically, the three conditions include essential amino acids (EAA), placebo, and control. The nutritional content from the dietary logs consisted of ____.

Variable	EAA	Placebo	Control	p	${\eta }_p^{\mathit{2}}$
Age (years)	21 ± 2	21 ± 3	22 ± 3	0.547	0.037
Height (cm)	180.0 ± 7.3	176.0 ± 4.3	179.1 ± 6.8	0.341	0.063
Weight (kg)	80.3 ± 8.9	77.7 ± 13.9	76.0 ± 11.0	0.618	0.029
Total calories	2541.0 ± 618.3	2343.1 ± 644.0	1962.9 ± 758.5	0.114	0.127
Protein (g)	168.8 ± 71.8	170.6 ± 93.7	134.1 ± 56.6	0.398	0.056
Carbohydrates (g)	243.5 ± 84.4	265.3 ± 95.8	170.7 ± 72.2	0.024 *	0.207
Fat (g)	94.1 ± 29.1	77.3 ± 18.7	78.3 ± 43.2	0.396	0.056
Protein (kcal)	670.0 ± 298.8	670.6 ± 385.3	539.0 ± 232.9	0.476	0.045
Carbohydrates (kcal)	948.5 ± 376.8	1061.0 ± 383.3	682.9 ± 288.7	0.037 *	0.186
Fat (kcal)	815.6 ± 295.0	695.7 ± 168.3	705.1 ± 388.5	0.582	0.033
Fiber (g)	13.7 ± 5.5	19.9 ± 5.8	19.0 ± 10.3	0.132	0.119
Vitamin A (mcg)	789.4 ± 1081.8	1159.7 ± 1882.6	545.2 ± 837.4	0.541	0.039
Vitamin C (mg)	41.4 ± 58.5	49.7 ± 51.2	75.5 ± 100.9	0.517	0.042
Calcium (mg)	1070.4 ± 1168.0	858.9 ± 979.1	821.2 ± 976.3	0.807	0.014
Iron (mg)	5.3 ± 3.1	4.6 ± 1.7	5.9 ± 3.2	0.624	0.030
Potassium (mg)	1773.3 ± 1039.9	2053.6 ± 1151.0	1597.4 ± 910.0	0.594	0.033
Sodium (mg)	3281.3 ± 1355.9	3156.8 ± 1329	2704.5 ± 1648.9	0.591	0.032

Abbreviations: cm: centimeters; kg: kilogram; g: gram; kcal: kilocalories; mcg: micrograms; mg: milligrams. *significantly different between groups.

3.2. Numerical pain scale ratings: best

There was a significant Condition × Time interaction (p < 0.001; ${\eta }_p^2$  = 0.268, Figure 1). Follow-up simple main effects of Time within each Condition indicated that the EAA group had greater NPRS Best ratings at 48 (25.1 ± 23.9 au) and 72 (18.3 ± 22.0 au) hours post EIMD relative to pre EIMD (0.7 ± 2.3 au) (p < 0.001; 95% CI: −38.8 to −10.1; g = 0.7 to 1.0; Prob = 97.8% to 99.2%), 48 and 72 hours post EIMD relative to immediately post EIMD (0 ± 0 au) (p < 0.001; 95% CI: −39.6 to −10.6; g = 0.8 to 1.0; Prob = 98.9% to 99.7%), and 48 hours post EIMD relative to 24 (6.3 ± 8.8 au) hours post EIMD (p = 0.001; 95% CI: −31.7 to −6.0; g = 0.8; Prob = 98.4%). For Placebo, there was greater NPRS Best ratings at 24 (13.1 ± 11.2 au), 48 (22.5 ± 17.8 au), and 72 (16.5 ± 12.0 au) hours post EIMD relative to pre EIMD (0 ± 0 au) (p < 0.001–0.008; 95% CI: −38.2 to −6.8; g = 1.1 to 1.3; Prob = 99.5% to 99.8% ) as well as 24, 48, and 72 hours post EIMD relative to immediately post EIMD (0 ± 0 au) (p < 0.001–0.008; 95% CI: −38.4 to −6.6; g = 1.1 to 1.3; Prob = 99.5% to 99.8%). For Control, there were no significant (p = 0.999) differences at any Time point. Follow-up simple main effects of Condition at each Time point indicated greater NPRS Best ratings at 24 hours post EIMD for Placebo (13.1 ± 11.2 au) relative to Control (0 ± 0 au) (p < 0.001; 95% CI: 5.0 to 21.2; g = 1.1; Prob = 99.6%), at 48 hours post EIMD for EAA (25.1 ± 23.9 au) and Placebo (22.5 ± 17.8 au) relative to Control (0.4 ± 1.4 au) (p = 0.002–0.009; 95% CI: 4.6 to 41.2; g = 1.0 to 1.1; Prob = 99.3% to 99.8%), and at 72 hours post EIMD for EAA (18.3 ± 22.0 au) and Placebo (16.5 ± 12.0 au) relative to Control (0.1 ± 0.3 au) (p = 0.007–0.025; 95% CI: 1.6 to 32.3; g = 0.8 to 1.2; Prob = 98.1% to 99.8%). There were no significant (p = 0.638–0.999) differences between Conditions at pre or immediately post EIMD.

Figure 1.

NPRS Best scores across time following EIMD. † greater than pre and post within condition; # placebo greater than control.

3.3. Numerical pain scale ratings: worst

There was a significant Condition × Time interaction (p < 0.001; ${\eta }_p^2$  = 0.429, Figure 2). Follow-up simple main effects of Time within each Condition indicated that the EAA group had greater NPRS Worst ratings immediately post EIMD (52.5 ± 20.8 au) as well as, 24 (31.4 ± 11.0 au), 48 (62.3 ± 25.2 au), and 72 (53.3 ± 22.6 au) hours post EIMD relative to pre EIMD (5.3 ± 8.3 au) (p < 0.001; 95% CI: −75.3 to −9.7; g = 1.9 to 2.5; Prob = 100%), immediately post EIMD relative to 24 hours post EIMD (p = 0.046; 95% CI: −42.0 to −0.2; g = 0.7; Prob = 99.4%), and 48 and 72 hours post EIMD relative to 24 hours post EIMD (p < 0.001–0.010; 95% CI: −51.7 to −3.6; g = 0.9 to 1.1; Prob = 99.2% to 99.8%). For Placebo, there was greater NPRS Worst ratings immediately post EIMD (58.0 ± 19.7 au) as well as, 24 (64.6 ± 24.6 au), 48 (69.5 ± 21.3 au), and 72 (64.5 ± 17.6 au) hours post EIMD relative to pre EIMD (10.8 ± 24.6 au) (p < 0.001; 95% CI: −78.6 to −33.4; g = 1.5 to 2.2; Prob = 100%). For Control, there were no significant (p = 0.999) differences at any Time point. Follow-up simple main effects of Condition at each Time point indicated greater NPRS Worst ratings immediately post EIMD for EAA (52.5 ± 20.8 au) and Placebo (58.0 ± 19.7 au) relative to Control (5.7 ± 4.3 au) (p < 0.001; 95% CI: −70.2 to −29.7; g = 1.9 to 2.6; Prob = 100%), at 24 hours post EIMD for Placebo (64.6 ± 24.6 au) relative to EAA (31.4 ± 11.0 au) and Control (5.6 ± 1.9 au) (p < 0.001; 95% CI: 16.0 to 75.7; g = 0.9 to 2.4; Prob = 99.9% to 100%) as well as EAA relative to Control (p < 0.001; 95% CI: 10.0 to 41.7; g = 1.4; Prob = 100%), at 48 hours post EIMD for EAA (62.3 ± 25.2 au) and Placebo (69.5 ± 21.3 au) relative to Control (4.8 ± 11.3 au) (p < 0.001; 95% CI: −85.6 to −37.6; g = 2.0 to 2.9; Prob = 100%), and at 72 hours post EIMD for EAA (53.3 ± 22.6 au) and Placebo (64.5 ± 17.6 au) relative to Control (6.5 ± 14.1 au) (p < 0.001; 95% CI: −76.8 to −29.0; g = 1.9 to 2.8; Prob = 100%). There were no significant (p = 0.933–0.999) differences between Conditions pre EIMD.

Figure 2.

NPRS Worst scores across time following EIMD. † greater than pre and post within condition; # placebo greater than control.

3.4. Numerical pain scale ratings: current

There was a significant Condition × Time interaction (p < 0.001; ${\eta }_p^2$  = 0.264, Figure 3). Follow-up simple main effects of Time within each Condition indicated that the EAA group had greater NPRS Current ratings immediately post EIMD (19.3 ± 17.8 au) as well as 24 (17.1 ± 11.2 au), 48 (34.0 ± 26.3 au), and 72 (26.8 ± 19.9 au) hours post EIMD relative to pre EIMD (1.0 ± 3.5 au) (p < 0.001-0.004; 95% CI: −50.4 to −3.8; g = 1.1 to 1.4; Prob = 99.8% to 100%) and at 48 hours relative to 24 hours post EIMD (p = 0.023; 95% CI: −32.3 to −1.5; g = 0.7; Prob = 95.6%). For Placebo, there were greater NPRS Current ratings immediately post EIMD (25.5 ± 11.2 au) as well as 24 (33.9 ± 22.6 au), 48 (42.0 ± 25.8 au), and 72 (29.5 ± 12.3 au) hours post EIMD relative to pre EIMD (1.5 ± 4.7 au) (p < 0.001; 95% CI: −59.5 to −14.5; g = 1.2 to 2.7; Prob = 100%). For Control, there were no significant (p = 0.999) differences at any Time point. Follow-up simple main effects of Condition at each Time point indicated greater NPRS Current ratings immediately post EIMD for EAA (19.3 ± 17.8 au) and Placebo (25.5 ± 11.2 au) relative to Control (0.9 ± 2.0 au) (p < 0.001–0.001; 95% CI: −37.0 to −6.7; g = 1.0 to 2.0; Prob = 99.8% to 100%), at 24 hours post EIMD for Placebo (33.9 ± 22.6 au) relative to EAA (17.1 ± 11.2 au) and Control (1.1 ± 2.1 au) (p < 0.001-0.021; 95% CI: 2.0 to 47.0; g = 0.5 to 1.4; Prob = 96.2% to 100%) as well as EAA relative to Control (p = 0.017; 95% CI: 2.4 to 29.5; g = 1.4; Prob = 100%), at 48 hours post EIMD for EAA (34.0 ± 26.3 au) and Placebo (42.0 ± 25.8 au) relative to Control (1.9 ± 5.8 au) (p < 0.001–0.001; 95% CI: −61.6 to −11.7; g = 1.2 to 1.4; Prob = 99.9% to 100%), and at 72 hours post EIMD for EAA (26.8 ± 19.9 au) and Placebo (29.5 ± 12.3 au) relative to Control (0.1 ± 0.3 au) (p < 0.001; 95% CI: −43.2 to −13.7; g = 1.2 to 2.2; Prob = 100%). There were no significant (p = 0.940–0.999) differences between conditions pre-EIMD.

Figure 3.

NPRS Current scores across time following EIMD. † greater than pre and post within condition; # placebo greater than control.

3.5. Numerical pain scale ratings: average

There was a significant Condition × Time interaction (p < 0.001; ${\eta }_p^2$  = 0.401). Follow-up simple main effects of Time within each Condition indicated that the EAA group had greater NPRS Average ratings immediately post EIMD (23.9 ± 9.0 au) as well as 24 (19.4 ± 15.9 au), 48 (40.5 ± 18.5 au), and 72 (32.8 ± 10.3 au) hours post EIMD relative to pre EIMD (2.3 ± 9.8 au) (p < 0.001; 95% CI: −52.6 to −7.9; g = 1.6 to 3.5; Prob = 100%), at 48 hours relative to immediately post EIMD and 24 hours post EIMD (p < 0.001–0.019; 95% CI: 1.8 to 34.8; g = 0.4 to 0.9; Prob = 96.9% to 99.1%), and at 72 hours relative to 24 hours post EIMD (p = 0.014; 95% CI: 1.8 to 25.1; g = 0.02; Prob = 97.3%). For Placebo, there were greater NPRS Average ratings immediately post EIMD (27.8 ± 9.0 au) as well as 24 (37.2 ± 15.9 au), 48 (44.7 ± 18.5 au), and 72 (36.8 ± 10.3 au) hours post EIMD relative to pre EIMD (4.1 ± 9.8 au) (p < 0.001; 95% CI: −56.4 to −17.9; g = 1.6 to 3.5; Prob = 100%) and at 48 hours relative to immediately post EIMD (p = 0.035; 95% CI: 0.7 to 32.9; g = 0.9; Prob = 98.6%). For control, there were no significant (p = 0.999) differences at any Time point. Follow-up simple main effects of condition at each time point indicated greater NPRS average ratings immediately post EIMD for EAA (23.9 ± 11.3 au) and placebo (27.8 ± 9.0 au) relative to control (2.2 ± 3.9 au) (p < 0.001; 95% CI: −34.4 to −13.4; g = 1.7 to 2.7; Prob = 100%), at 24 hours post EIMD for Placebo (37.2 ± 15.9 au) relative to EAA (19.4 ± 4.5 au) and control (2.2 ± 0.6 au) (p < 0.001; 95% CI: 8.0 to 44.5; g = 0.9 to 2.1; Prob = 99.7% to 100%) as well as EAA relative to Control (p < 0.001; 95% CI: 8.1 to 26.2; g = 2.8; Prob = 100%), at 48 hours post EIMD for EAA (40.5 ± 23.6 au) and Placebo (44.7 ± 18.5 au) relative to control (2.4 ± 6.7 au) (p < 0.001; 95% CI: −60.2 to −21.1; g = 1.6 to 2.1; Prob = 100%), and at 72 hours post EIMD for EAA (32.8 ± 19.2 au) and Placebo (36.8 ± 10.3 au) relative to Control (2.2 ± 4.7 au) (p < 0.001; 95% CI: −47.8 to −18.1; g = 1.6 to 3.0; Prob = 100%). There were no significant (p = 0.883–0.999) differences between Conditions pre EIMD.

3.6. Pain pressure threshold

There was no significant Condition × Time interaction (p = 0.862; ${\eta }_p^2$  = 0.023, Figure 4) or main effects of Condition (p = 0.546; ${\eta }_p^2$  = 0.036). There was, however, a significant main effect of Time (p = 0.018; ${\eta }_p^2$  = 0.101). Specifically, collapsed across Condition, PPT decreased from immediately post EIMD (15.0 ± 7.3 kgf) to 48 (12.8 ± 7.0 kgf) hours post EIMD (p = 0.019; 95% CI: 0.2 to 4.1; g = 0.5; Prob = 90.3%).

Figure 4.

PPT across time following EIMD. * 48 hours significantly lower than post.

3.7. Calf muscle thickness

There was a significant Condition × Time interaction (p < 0.001; ${\eta }_p^2$  = 0.463, Figure 5). Follow-up simple main effects of Time within each Condition indicated that the EAA group had greater increases in Calf Muscle Thickness immediately post EIMD (1.82 ± 0.4 cm) as well as 24 (1.83 ± 0.3 cm) and 48 (1.79 ± 0.2 cm) hours post EIMD relative to pre EIMD (1.66 ± 0.3 cm) (p < 0.001–0.032; 95% CI: −0.2 to −0.01; g = 0.9 to 1.3; Prob = 83% to 86%), however, there were no significant differences at 72 (1.78 ± 0.3 cm) hours post EIMD relative to pre EIMD (p = 0.062). For Placebo, there were greater increases in Calf Muscle Thickness immediately post EIMD (2.0 ± 0.4 cm) as well as 24 (2.0 ± 0.3 cm), 48 (2.07 ± 0.4 cm), and 72 (2.04 ± 0.4 cm) hours post EIMD relative to pre EIMD (1.63 ± 0.2 cm) (p < 0.001; 95% CI: −0.6 to −0.2; g = 1.4 to 1.9; Prob = 98.2% to 99.2%). For Control, there were no significant (p = 0.999) differences at any Time point. Follow-up simple main effects of Condition at each Time point indicated greater Calf Muscle Thickness at 48 hours post EIMD for Placebo (2.06 ± 0.4 cm) relative to Control (1.68 ± 0.2 cm) (p = 0.004; 95% CI: −0.7 to −0.1; g = 0.9; Prob = 99%) as well as 72 hours post EIMD for Placebo (2.04 ± 0.4 cm) relative to Control (1.68 ± 0.1 cm) (p = 0.031; 95% CI: −0.7 to −0.03; g = 0.8; Prob = 97.9%). There were no significant (p = 0.065–0.999) differences between Conditions at pre EIMD, immediately post EIMD, and 24 hours post EIMD.

Figure 5.

Calf muscle thickness across time following EIMD. * EAA greater than pre at post, 24, and 48 hours; # placebo greater than pre at post, 24, 48, and 72 hours; † greater than control; ‡ greater than EAA.

3.8. Achilles tendon thickness

There was no significant Condition × Time interaction (p = 0.229; ${\eta }_p^2$  = 0.081, Figure 6). There were, however, significant main effects of Condition (p = 0.003; ${\eta }_p^2$  = 0.310) and Time (p = 0.005; ${\eta }_p^2$  = 0.135). Specifically, collapsed across Time, Achilles Tendon Thickness was greater for Control (0.41 ± 0.1 cm) relative to EAA (0.36 ± 0.04 cm) and Placebo (0.35 ± 0.03 cm) (p = 0.003–0.038; 95% CI: 0.01 to 0.1; g = 0.6 to 1.0; Prob = 100%). Collapsed across Condition, Achilles Tendon Thickness decreased from pre EIMD (0.39 ± 0.05 cm) to immediately post EIMD (0.36 ± 0.1 cm) (p < 0.001; 95% CI: 0.01 to 0.04; g = 0.8; Prob = 97.7%).

Figure 6.

Achilles tendon thickness across time following EIMD. * post significantly lower than pre.

3.9. Calf circumference

There was a significant Condition × Time interaction (p < 0.001; ${\eta }_p^2$  = 0.416, Figure 7). Follow-up simple main effects of Time within each Condition indicated that the EAA group had greater increases in Calf Circumference immediately post EIMD (37.7 ± 2.4 cm) as well as 24 (37.1 ± 2.5 cm), 48 (37.1 ± 2.4 au), and 72 (37.1 ± 2.5 au) hours post EIMD relative to pre EIMD (36.6 ± 2.4 cm) (p < 0.001-0.043; 95% CI: −1.5 to −0.01; g = 0.6 to 2.2; Prob = 64.6% to 84%). Also, for EAA, Calf Circumference was greater immediately post EIMD relative to 24, 48, and 72 hours post EIMD (p < 0.001; 95% CI: 0.2 to 1.5; g = 1.0 to 1.7; Prob = 72.2% to 72.5%). For Placebo, there was greater increases in Calf Circumference immediately post EIMD (37.2 ± 2.2 cm) as well as 24 (36.6 ± 2.2 cm), 48 (36.8 ± 2.1 au), and 72 (36.5 ± 2.3 au) hours post EIMD relative to pre EIMD (35.9 ± 2.3 cm) (p < 0.001–0.007; 95% CI: −1.7 to −0.1; g = 1.5 to 2.7; Prob = 69.1% to 86.1%). Also, for Placebo, Calf Circumference was greater immediately post EIMD relative to 24, 48, and 72 hours post EIMD (p < 0.001–0.010; 95% CI: 0.08 to 1.7; g = 0.9 to 1.6; Prob = 65.8% to 72.7%). For Control, there were no significant (p = 0.999) differences at any Time point. Follow-up simple main effects of Condition at each Time point indicated no significant (p = 0.139–0.999) differences between Conditions at any Time point.

Figure 7.

Calf circumference across time following EIMD. * EAA greater than pre at post, 24, 48, and 72 hours; ^ EAA post greater than 24, 48, and 72 hours; # placebo greater than pre at post, 24, 48, and 72 hours and post greater than 24, 48, and 72 hours.

4. Discussion

The purpose of this study was to investigate the effects of EAA supplementation on indirect markers of musculotendinous recovery following eccentric plantar flexor exercise. The eccentric protocol elicited several responses consistent with EIMD, including increases in perceived pain, elevations in calf muscle thickness and circumference, and reductions in pain pressure threshold across the recovery period, indicating localized musculotendinous stress and inflammatory responses typical of eccentric loading. Importantly, EAA supplementation attenuated perceptual responses during the early recovery period, with lower soreness ratings compared to placebo at 24 hours post-exercise. Additionally, while both exercise conditions demonstrated increases in calf muscle thickness, values in the EAA group returned to baseline by 72 hours, whereas elevations persisted in the placebo group, suggesting a more rapid resolution of muscle swelling with EAA supplementation. Collectively, these findings indicate that EAA supplementation may enhance aspects of early recovery following eccentric exercise, particularly by reducing perceived soreness and accelerating the resolution of muscle swelling.

The increases in numerical pain ratings across all NPRS categories suggest that the eccentric plantar flexor protocol successfully elicited delayed onset muscle soreness (DOMS). Participants in both exercise conditions demonstrated substantial elevations in perceived soreness immediately following exercise that persisted throughout the 24–72 h recovery period. This pattern is consistent with the established progression of DOMS following eccentric exercise, where soreness typically develops within the first 24 hours and peaks between 24 and 48 hours as a result of structural disruption of muscle fibers, inflammatory signaling, and nociceptor sensitization within the musculotendinous unit [2,31,32]. Similar soreness responses were observed in our previous investigation using a comparable eccentric plantar flexor protocol, which demonstrated large increases in perceived pain during the early recovery period following EIMD [5].

Additionally, the present findings indicate that EAA supplementation may attenuate the perception of soreness during the early phase of recovery. Specifically, the EAA group reported substantially lower pain ratings than the placebo condition at 24 hours post-exercise. Also, worst pain ratings at 24 hours were approximately 51% lower in the EAA group compared with placebo. Similarly, current pain ratings were ~50% lower in the EAA condition relative to placebo, and average pain ratings were ~48% lower. Despite these early differences, soreness remained elevated in both exercise conditions at 48 and 72 hours post-exercise. One potential explanation for this effect is the rapid availability of EAAs following exercise, particularly leucine, which is known to stimulate MPS through activation of the mTOR signaling pathway [14,33–35]. Increased amino acid availability may accelerate early repair processes within damaged muscle fibers and potentially reduce the magnitude of inflammatory signaling associated with nociceptor activation [14,33,34]. Previous research examining amino acid supplementation following damaging exercise has produced mixed findings regarding its influence on soreness and recovery. For example, Szych et al. [18]. demonstrated that acute ingestion of EAAs (6.6 g/day) during repeated aerobic and resistance exercise reduced symptoms of DOMS while also improving several performance measures in previously sedentary participants. Similarly, Nosaka et al. [36] reported that supplementation with a mixture of essential and non-essential amino acids attenuated muscle soreness and reduced circulating markers of muscle damage during the recovery period following eccentric elbow flexor exercise when supplementation was maintained for several days post-exercise. In contrast, Vieillevoye et al. [20] observed that although eccentric bench press exercise induced substantial increases in soreness and circulating creatine kinase and myoglobin, EAA supplementation had minimal effects on perceived muscle soreness and did not significantly alter the time-course of recovery. Together, these findings suggest that while amino acid availability may influence aspects of muscle repair and recovery following eccentric exercise, its effects on perceptual markers of soreness appear to depend on factors such as supplementation timing, dosage, exercise modality, and the duration of the recovery period. It should also be noted that the citations supporting the mTOR-mediated mechanistic explanation above are intended as rationale rather than as evidence that this pathway was directly assessed in the present study; citations 11 and 12 provide more direct mechanistic support for leucine-mediated mTOR activation than some of the sources currently cited.

The observed decrease in PPT from immediately post-exercise to 48 hours post-exercise further supports that the eccentric exercise protocol produced localized musculotendinous changes consistent with EIMD. PPT primarily reflects localized mechanical nociception within the muscle tissue, which is largely mediated by sensitization of group III and IV afferent nerve endings and inflammatory signaling following structural disruption of muscle fibers [37,38]. Previous work has shown that activation of peripheral excitatory amino acid receptors can directly reduce PPT and increase mechanical allodynia in skeletal muscle, highlighting the role of local nociceptive signaling in pressure sensitivity responses [39]. In contrast, nutritional interventions such as EAA supplementation are more likely to influence systemic processes related to muscle protein turnover, inflammation, and tissue remodeling rather than directly altering nociceptor sensitivity to mechanical pressure [40]. Thus, although the eccentric protocol produced measurable changes in local mechanical sensitivity, EAA supplementation did not appear to meaningfully alter this response within the 72-hour recovery period. It is also worth noting that some of the cited evidence regarding group III/IV afferent sensitization is derived from central sensitization literature, and its direct applicability to the peripheral EIMD response described here should be considered when this section is finalized.

Following the eccentric protocol, calf muscle thickness and circumference increased, further supporting that the exercise bout induced localized structural responses consistent with EIMD. Eccentric loading is known to disrupt sarcomeric integrity and increase membrane permeability, which contributes to inflammatory cell infiltration, interstitial fluid accumulation, and transient swelling within the exercised musculature [2,36]. In the present study, both the EAA and placebo conditions demonstrated significant increases in calf muscle thickness immediately post-exercise and during the subsequent recovery period. This pattern is consistent with previous work demonstrating that eccentric plantar flexor exercise produces measurable increases in gastrocnemius thickness, likely reflecting edema and localized inflammatory responses during the early stages of recovery [5]. In our previous plantar flexor study, gastrocnemius thickness increased by 22.4% immediately post-exercise and remained elevated by 12.9% at 72 hours, highlighting muscle thickness as one of the more sensitive structural markers of eccentric EIMD [5].

Interestingly, the recovery pattern differed slightly, whereby the EAA group demonstrated increases in calf muscle thickness immediately post-exercise and at 24 and 48 hours, but the values were no longer elevated by 72 hours. For placebo, these responses remained elevated through 72 hours. Although between-group differences were not significant at earlier time points, the persistence of elevated muscle thickness only in the placebo condition may suggest an accelerated recovery of exercise-induced swelling in the EAA group. Eccentric exercise disrupts sarcomeric structures and increases membrane permeability, leading to inflammatory cell infiltration and interstitial fluid accumulation within the muscle during the early stages of recovery. Increased availability of EAAs, particularly leucine, may support repair of myofibrillar proteins through stimulation of MPS via mTOR signaling; however, MPS and mTOR activity were not directly measured in the present study, and this remains a proposed rationale for the observed swelling pattern rather than a demonstrated mechanism. Consistent with this rationale, Matsui et al. [17]. reported that supplementation with a leucine-enriched EAA mixture attenuated peak creatine phosphokinase concentrations approximately 5 days following eccentric elbow flexor exercise, suggesting reduced muscle damage during recovery. Similarly, a recent meta-analysis [41] reported that branched-chain amino acid supplementation significantly reduced creatine kinase concentrations immediately and 72 hours following EIMD and attenuated DOMS 24–96 hours post-exercise. These findings suggest that increased amino acid availability may contribute to improved recovery of muscle tissue integrity following damaging exercise.

Calf circumference demonstrated a similar pattern of swelling with no differences between the groups. Both the EAA and placebo groups exhibited significant increases immediately following exercise that remained elevated through 72 hours. Limb circumference is commonly used as an indirect marker of post-exercise edema and fluid accumulation following EIMD [5]. A recent systematic review evaluating indirect markers of EIMD identified limb circumference as one of the commonly reported indicators of post-exercise swelling, although it is used less frequently than markers such as strength loss or soreness [42]. However, unlike muscle thickness, no between-condition differences were observed for circumference at any time point. This difference in responses likely reflects the lower sensitivity of circumference measurements, which capture global limb size and may be influenced by fascia, skin, and subcutaneous tissue rather than intramuscular swelling alone. In contrast, ultrasound-derived assessments of muscle thickness provide a localized assessment of structural changes within the muscle. Previous work has similarly demonstrated that ultrasound-derived muscle thickness can detect localized changes in muscle swelling following exercise that may not be reflected in broader measures of body size or fluid distribution [43]. Evidence examining the influence of amino acid supplementation on limb swelling is also limited and inconsistent. For example, Howatson et al. [44] reported that BCAA supplementation reduced several markers of muscle damage but did not influence thigh or calf circumference measured at 24–96 hours following a damaging bout of drop jumps. Conversely, Ra et al. [45] observed modest reductions in upper-arm circumference across the 4-day recovery period when BCAA were consumed prior to eccentric exercise. Together, these findings suggest that limb circumference may be relatively insensitive to nutritional interventions targeting muscle recovery. Thus, while both circumference and ultrasound confirmed that the eccentric protocol induced localized swelling, the ultrasound findings in the present study may better capture subtle differences in intramuscular recovery that are not detectable using broader anthropometric measures.

Achilles tendon thickness demonstrated a different response pattern than the muscle-derived markers, with a reduction from pre-exercise to immediately post-exercise. Acute decreases in tendon thickness following mechanical loading have been previously reported and are generally attributed to transient fluid redistribution within the tendon matrix during high mechanical strain [46]. Eccentric loading can increase intratendinous hydrostatic pressure and promote fluid movement out of the tendon's extracellular matrix, resulting in temporary reductions in tendon cross-sectional dimensions [5,6]. Similar acute reductions in Achilles tendon thickness following eccentric plantar flexor exercise have been previously observed, suggesting that this response reflects normal biomechanical behavior of tendon tissue under load rather than structural damage [5]. Indeed, the EAA supplementation did not influence these acute tendon responses. This finding is consistent with the current literature, as most evidence regarding EAAs and EIMD is derived from skeletal muscle rather than tendon tissue [10,17,18]. Although EAA or leucine-enriched EAA supplementation has been shown to modestly improve recovery of muscle damage markers such as creatine kinase and soreness [18], direct evidence demonstrating acute effects on tendon structure or collagen remodeling following damaging exercise is limited and largely indirect. Consequently, while EAA supplementation may influence muscle recovery processes, its effects on acute tendon responses following eccentric loading remain unclear.

Several limitations should be considered when interpreting these findings. First, the sample consisted exclusively of young recreationally active males, which limits the generalizability of the results to females, older adults, or highly trained populations. Second, the recovery period was limited to 72 hours following the damaging exercise bout, which may not fully capture longer-term recovery processes associated with muscle remodeling and repair. Third, the study relied on indirect markers of musculotendinous recovery and did not include circulating biomarkers of muscle damage or direct measures of MPS. The study also did not include a functional performance measure, such as maximal voluntary contraction or plantar flexor torque, and therefore cannot confirm whether the eccentric protocol produced a functional impairment consistent with muscle damage. Finally, dietary intake was assessed using self-reported logs, which may introduce reporting inaccuracies and variability in nutritional intake across participants. Fifth, because the placebo comparator was maltodextrin rather than a whole-protein source, the present findings can only be interpreted as evidence for EAA supplementation relative to a non-amino-acid control and do not support conclusions regarding any advantage of free-form EAAs over intact protein.

In summary, the eccentric plantar flexor protocol induced responses consistent with EIMD, including increased soreness, reductions in pain pressure threshold, and transient increases in muscle swelling as evidenced by ultrasound-derived calf muscle thickness and limb circumference. Ultrasound measures indicated the presence of localized edema following exercise, which is commonly observed after eccentric loading due to inflammatory responses and fluid accumulation within damaged muscle tissue. EAA supplementation modestly reduced perceived soreness during the early recovery phase and was associated with a faster recovery of ultrasound-derived muscle thickness, with swelling returning to baseline by 72 hours in the EAA group but remaining elevated in the placebo condition. However, supplementation did not meaningfully influence PPT or Achilles tendon thickness. Overall, these findings suggest that EAA supplementation may provide modest benefits during early recovery by reducing soreness and accelerating the recovery of muscle swelling following eccentric exercise, although it does not appear to substantially influence tendon morphology within 72 hours following EIMD.

Funding Statement

This study was supported by Optimal Labs, LLC. The company provided the EAA supplements used in the study and financial support for participant compensation. The sponsor had no role in data collection, statistical analyzes, interpretation of the results, manuscript preparation, or decision to publish.

Disclosure statement

Ecaterina Vasenina serves as a scientific advisor for Legion Athletics. This relationship had no role in the design, execution, analysis, or interpretation of the study results. The remaining authors declare no conflicts of interest.