Abstract
ves: This study investigates the effects of 600 mg/day Ashwagandha root extract on physiological stress biomarkers, perception of recovery, muscle strength and aerobic capacity in team sports athletes during pre-season training, a period associated with ele- vated cortisol and accumulated training stress.Methods: Fifty-six athletes (26.8±4.4 years, 1.74±0.10 m, 79.4±17.3 kg, 11.0±7.1 career years) across rugby, water polo and football were randomly assigned to an Ashwagandha (ASH;n= 28, 14 males and 14 females) or placebo (PLA;n= 28, 14 males and 14 females) group for 42 days. Salivary biomarkers were assessed after training, muscle strength and aerobic capacity were measured during train- ing, and perception of recovery was evaluated with Hooper Index (HI) the following day. Mixed ANOVA was used to determine group×time interactions and Bonferroni post hoc analyses were conducted for multiple pairwise comparisons.Results: In female athletes, salivary cortisol increased significantly in PLA (p= 0.001), while recovery parameters such as the overall HI score (p= 0.001), Delayed
were measured during train- ing, and perception of recovery was evaluated with Hooper Index (HI) the following day. Mixed ANOVA was used to determine group×time interactions and Bonferroni post hoc analyses were conducted for multiple pairwise comparisons.Results: In female athletes, salivary cortisol increased significantly in PLA (p= 0.001), while recovery parameters such as the overall HI score (p= 0.001), Delayed Onset Muscle Soreness (DOMS) (p= 0.008) and perception of fatigue (p= 0.026) scores improved significantly in ASH. In males, salivary cortisone increased significantly in PLA (p= 0.022), while Countermovement Jump (CMJ) improved significantly in ASH (p= 0.018). Pull-up performance increased in both PLA (p= 0.004) and ASH (p< 0.0001) in males.Conclusions: Supplementation with 600 mg/day of Ashwagandha root extract for 42 days may stabilise stress biomarkers, improve percep- tion of recovery and enhance muscle strength in team sports athletes during pre-season training. The trial is registered on ClinicalTrials.gov with the ID NCT07041853. Keywords:ashwagandha; athletes; pre-season; muscle strength; aerobic capacity; recovery; stress; cortisol; cortisone; randomised controlled trial 1. Introduction Pre-season training is a pivotal phase in an athlete’s annual cycle, aimed at developing physical capacities, refining technical skills and preparing the body for the competitive Nutrients2026,18, 230 https://doi.org/10.3390/nu18020230
Nutrients2026,18, 230 2 of 22 in-season [1]. The cumulative load of pre-season training has been associated with changes in cortisol concentrations [2,3], which may impair recovery, disrupt anabolic processes and increase the risk of overtraining or injury [4]. In contact and team sports, these risks are further amplified by the intermittent high-intensity nature of training schedule demands, which often include sprints, collisions and rapid directional changes [5–7]. These demands can place strain on both the neuromuscular and endocrine systems, influencing cortisol response [8]. Physiological stress can be evaluated through salivary measurements of cortisol and cortisone: hormones that indicate activity of the hypothalamic–pituitary– adrenal (HPA) axis [9]. Moderate- to high-intensity exercise can provoke increases in circulating cortisol levels [10]. In a study involving female athletes, participation in high- stakes matches was associated with a marked reduction in sleep quality, which coincided with a substantial 354% increase in salivary cortisol concentrations [11]. Elevated post- exercise cortisol levels have been linked to suppressed immune function, increased muscle protein breakdown and disrupted sleep patterns, all of which can impair performance and recovery [12,13]. Consequently, effective recovery strategies during the pre-season are essential to miti- gate the impact of elevated physiological stress responses and promote optimal adaptation to training. Typical recovery strategies for athletes involve achieving adequate sleep quality, efficient muscle repair and the restoration of hormonal balance [14,15]. Monitoring per- ceived recovery can offer valuable insight into an athlete’s readiness to train and compete, encompassing perceptions of stress, fatigue, muscle soreness and sleep quality. Assessing salivary hormones in addition may enable practitioners to better evaluate an athlete’s physiological and psychological adaptation throughout the pre-season phase. Ashwagandha (Withania somnifera) root extract, a traditional adaptogenic herb, is known for its potential to modulate the stress response, enhance physical performance and improve the recovery process in healthy populations [16,17]. Recent systematic reviews and meta-analyses have highlighted the efficacy of Ashwagandha supplementation in clinical trials in reducing systemic stress by having a positive effect on wellbeing, muscle strength and exercise endurance parameters [18,19]. Trials using standardised root extracts have demonstrated significant reductions in cortisol in healthy adults and
enhance physical performance and improve the recovery process in healthy populations [16,17]. Recent systematic reviews and meta-analyses have highlighted the efficacy of Ashwagandha supplementation in clinical trials in reducing systemic stress by having a positive effect on wellbeing, muscle strength and exercise endurance parameters [18,19]. Trials using standardised root extracts have demonstrated significant reductions in cortisol in healthy adults and individuals experienc- ing mild to moderate stress, with effects observed across a range of doses (125–600 mg/day) and supplementation periods (4–12 weeks). These studies indicate that Ashwagandha may modulate the HPA axis as its mechanism of action by reducing the circulating cortisol and potentially improving stress resilience and sleep quality as a result [20,21]. The bioactive constituents of Ashwagandha include steroidal flavanol glycosides, gly- cowithanolides, steroidal lactones and phenolics, with more than 12 alkaloids, around 40 withanolides and several sitoindosides reported across its roots, aerial parts and berries [22]. The root extract of the Ashwagandha plant is associated with lower risk of containing, or having negligible levels of, potentially harmful compounds such as with- anone and withaferin A that are found in higher content in the leaf of the herb [23–25]. These compounds may induce liver toxicity or adverse reactions with overuse [26], despite having been reported to elicit anticarcinogenic effects [27–29]. While it is not established, this may explain the adverse results found in research documenting liver injuries following Ashwagandha use [30,31]; the supplements reported are shown to contain the leaf or do not state the amount of withanolides, which are the active compounds that produce the adaptogenic effects. A recentin vitrostudy using primary human hepatocytes found that ethanolic Ashwagandha leaf extract exhibited dose- and time-dependent hepatocellular toxicity, suggesting possible liver risks at higher concentrations. In contrast, the root extract modulated CYP3A4 activity (an enzyme integral to drug metabolism, metabolising ap- proximately 30–50% of known drugs [32]), indicating potential drug–herb interactions [33]. https://doi.org/10.3390/nu18020230
Nutrients2026,18, 230 3 of 22 This suggests individuals taking prescription medications should avoid using it, echoed in a case study that found a patient having involuntary muscle contractions following Ashwagandha supplementation alongside use of 2.5 mg diazepam [34]. However, the safety profile of root-extracted Ashwagandha with a standardised amount of withanolides has been well-documented, with studies supporting its use in healthy participants [35–38], with one study observing administration over 12 months [39]. Therefore, the chosen in- vestigational product KSM-66 Ashwagandha was obtained from the manufacturer Ixoreal Biomed Inc., Los Angeles, CA, USA. KSM-66 is a commercially available Ashwagandha extract derived exclusively from the root and standardised to the highest concentration. The supplement is produced through a green chemistry method that is devoid of any alcohol or chemical solvents. The product contains a root-only extract from Ashwagandha with >5% of withanolides as estimated by the HPLC method. There is a lack of research specifically examining the effects of Ashwagandha sup- plementation in athletes participating in contact and team sports. Team sports athletes experience unique pre-season demands, including repeated high-intensity efforts and rapid changes in direction, which may influence recovery and performance [40]. This popula- tion is therefore suitable for evaluating interventions aimed at supporting adaptation and resilience during periods of elevated training stress. The optimal dosage, duration and timing of supplementation remain subjects of ongoing investigation. This study aims to address these gaps by evaluating the effects of 600 mg/day of root-extracted Ashwagandha over a 42-day period on salivary hormone concentrations, as well as perceived recov- ery and muscle strength in the pre-season period in male and female semi-professional athletes assigned to an academy. Accordingly, the present study examines the effects of Ashwagandha root extract supplementation on physiological stress biomarkers, perceived recovery and muscle strength across the pre-season period in athletes, with the aim of assessing whether an herbal supplement can improve these areas. It is hypothesised that root extract Ashwagandha supplementation, across male and female team sports ath- letes, will achieve the following: (1) reduce salivary cortisol and cortisone concentrations, (2) improve recovery perception, and (3) enhance muscle strength outcomes,
recovery and muscle strength across the pre-season period in athletes, with the aim of assessing whether an herbal supplement can improve these areas. It is hypothesised that root extract Ashwagandha supplementation, across male and female team sports ath- letes, will achieve the following: (1) reduce salivary cortisol and cortisone concentrations, (2) improve recovery perception, and (3) enhance muscle strength outcomes, providing a clear, testable framework for assessing its effectiveness in a team sport setting. 2. Materials and Methods 2.1. Study Design The study followed a randomised, double-blind, placebo-controlled design over a 6-week period. Participants were recruited through convenience sampling from a sports academy in Barcelona, Spain and included athletes from rugby, water polo and football. Eligible participants were team sports athletes assigned to a professional sports academy in Barcelona, Spain, aged above 18 years, competing at a sub-elite level and classified as ‘healthy’ or free from disease. Exclusion criteria included active supplemen- tation with other ergogenic aids, pregnancy, allergies to nightshades, use of medications or hormonal contraceptives, to avoid potential interactions of Ashwagandha root extract, which may modulate CYP3A4 enzyme activity involved in drug metabolism [33]. With- drawal from the study was permitted for participants who requested to exit or failed to complete the required study tests. Participants received a small monetary compensation of EUR 20 per testing session to support compliance and retention throughout the study period. Offering payment to research participants is recognised as an ethically acceptable method of enhancing recruitment and compliance in clinical trials [41]. Participants were randomised using an online (https://www.randomizer.org/ randomisation allocation programme to either the Ashwagandha or placebo group, with equal distribution by sex. The study participants (n= 56) were between the ages of 18 and https://doi.org/10.3390/nu18020230
Nutrients2026,18, 230 4 of 22 35 years (26.8±4.4 years, 1.74±0.10 m, 79.4±17.3 kg and 11.0±7.1 years in career) (Table). Table 1.Participant characteristics and differences by study group. ASH (n= 28) PLA (n= 28) p-Value Female (n= 28) Male (n= 28) Age (years) 26.3 (5.1) 27.3 (3.5) 0.400 27.5 (4.2) 26.1 (4.5) Height (cm) 175.8 (10.2) 173.4 (9.8) 0.382 166.8 (4.7) 182.3 (7.5) Body mass (kg) 81.8 (14.5) 76.9 (18.3) 0.280 66.4 (9.6) 92.3 (10.9) Career (years) 9.6 (6.6) 12.4 (7.4) 0.149 7.3 (6.5) 14.6 (5.8) ASH = 600 mg daily dose of Ashwagandha root extract; PLA = placebo. Significance values were calculated using independentt-tests between groups ASH and PLA and Female and Male. 2.2. Procedures At the first visit, all participants received detailed information about the study proce- dures and were provided with written informed consent. Information about self-reported height and mass and career length of each athlete was collected from the strength and conditioning staff members. The study timeline is presented in Figure. For each of the testing sessions, all participants completed assessments evaluating salivary hormone mark- ers, muscle strength, aerobic capacity and perception of recovery using a questionnaire the following day. Data collection for both timepoints took place at the gym of the academy, which is a familiar training ground for all participants. Figure 1.Study timeline. CMJ, countermovement jump; HI, Hooper Index. Training load was recorded by documenting the type, frequency and duration of each session within the academy programme. Sessions combined resistance and conditioning work typical of pre-season preparation with comparable overall demands across sports despite minor differences in exercise content. Strength and conditioning staff supervised all sessions, recording the activities performed, their duration and athlete completion. Each session was designed to elicit a physiological training response consistent with pre-season https://doi.org/10.3390/nu18020230
Nutrients2026,18, 230 5 of 22 intensity. Training sessions commenced at 19:30 h and there was a consistent temperature of 28–30 ◦ C with 70–80% humidity. Saliva samples were collected at the end of every training session using Salivette sam- ple tubes (Sarstedt, Nümbrecht, Germany), documented as a reliable method to evaluate salivary cortisol compared to total and calculated serum cortisol levels [42,43]. Participants were instructed to not eat or drink (except water) at least 30 min prior to sampling. At 21:00 h, each athlete provided a passive saliva sample. Samples were immediately stored on ice before being transferred to a−20 ◦ C freezer for later analysis of hormonal markers. The salivary samples had to be equal to or above 0.5 mL in quantity in order to evaluate cortisol, cortisone, testosterone, DHEA-S and amylase. Stress biomarkers were measured post-training to monitor the acute hormonal response to resistance exercise, as they play a key role in tissue remodelling and adaptation [44]. Assessing cortisol in particular, the primary variable of this study, allows for the quantification of exercise-induced physi- ological stress and provides insight into the effectiveness and recovery demands of the training protocol. Recovery and wellbeing were assessed using the Hooper Index (HI), a validated sub- jective tool designed to capture exercise-induced fatigue in athletes [45]. The HI evaluates four key components: sleep quality, stress, fatigue and muscle soreness, with participants rating each domain on a 1–10 scale, where lower scores indicate better recovery. This method provides a practical and sensitive measure of how athletes perceive their recovery following training. To standardise data collection, the HI was distributed via WhatsApp and completed at 12:00 h on the day following each training session. Menstrual cycle data were self-reported but excluded from analysis. Evidence suggests hormonal fluctuations have minimal effects on strength, performance or recovery, and inconsistent verification methods limit firm conclusions [46]. Although small effects on recovery have been observed in endurance athletes, the menstrual cycle appears to be one of several minor stressors rather than a key factor [47,48]. Maximal strength was determined at the start of the training session with handgrip
suggests hormonal fluctuations have minimal effects on strength, performance or recovery, and inconsistent verification methods limit firm conclusions [46]. Although small effects on recovery have been observed in endurance athletes, the menstrual cycle appears to be one of several minor stressors rather than a key factor [47,48]. Maximal strength was determined at the start of the training session with handgrip strength test, measured with the dominant hand using a dynamometer (CAMRY, CA, USA). It is a commonly used tool in sports performance and demonstrates validity and reliability in relation to athlete strength [49]. The validity for the use of a CAMRY dy- namometer is documented [50] when comparing against the Jamar dynamometer. Only one maximal attempt was required, as previous work has shown single trials to yield results comparable to the mean of three [51]. Grip span was adjusted individually for comfort and accuracy. Following this measure, participants performed one-repetition maximum (1RM) tests for squat, bench press, deadlift and power clean. Additionally, explosive performance was captured through pull-ups, countermovement jump (CMJ) and standing broad jump These tests are well-established as reliable field indicators of lower-limb power [52]. Aerobic fitness was measured with the Bronco test, which consists of a 1.2 km shuttle run test. The Bronco test shows good validity, with maximum heart rate significantly correlated to match-play heart rate [53]. Testing order was not fixed between participants. After the initial baseline measurements, participants commenced a 42-day daily sup- plementation protocol, receiving either 600 mg/day of the KSM-66 Ashwagandha-root extract (ASH), standardised to >5% withanolides, or 600 mg/day of chickpea flour encap- sulated in hydroxypropyl methylcellulose (HPMC) as a placebo (PLA). Researchers were blinded to the allocation. Supplements were taken with dinner and a glass of water, as instructed by the study investigators. Chickpea flour was chosen for its visual similarity to Ashwagandha extract and its gluten-free properties, accommodating participants with potential gluten intolerance. The KSM-66 Ashwagandha extract is certified by Informed https://doi.org/10.3390/nu18020230
the study investigators. Chickpea flour was chosen for its visual similarity to Ashwagandha extract and its gluten-free properties, accommodating participants with potential gluten intolerance. The KSM-66 Ashwagandha extract is certified by Informed https://doi.org/10.3390/nu18020230
Nutrients2026,18, 230 6 of 22 Ingredient, a comprehensive anti-doping programme that tests raw materials for banned substances in dietary supplements. Supplement adherence throughout the study was 100% in the remaining participants, monitored through daily check-ins conducted by the research team and academy staff. The monitoring of adverse events was undertaken jointly by the academy staff and research team. In addition, a supplement satisfaction survey was taken at the end of the supplementation period in both the ASH and PLA groups. Scores were taken from a Likert-scale-style survey (1 = very satisfied, 10 = very dissatisfied), inspired by the previous literature that assessed supplement satisfaction of tart cherry after analysing for muscle pain during running [54]. Figure of Reporting Trials) flow diagram of the study. Figure 2.CONSORT diagram. Consolidated Standards of Reporting Trials (CONSORT) diagram. ASH, root extract of Ashwagandha; PLA, placebo. 2.3. Ethical Committee The study and the details of the informed consent form were approved by the Research Ethics Committee of the School of Health Sciences of Blanquerna Institute, University Ramon Llull (CER-FCSB) in June 2025 (Approval number: 05-03-2025). The trial was registered prior to data collection on ClinicalTrials.gov with ID NCT07041853. 2.4. Statistical Analysis The required sample size for the study was estimated using G*Power (version 3.1.9.6) and follows the guidelines for sample size estimation in sport and exercise science research by including a formal a priori sample size estimation and rationale [55]. Previous research shows Ashwagandha produces a large effect on symptoms of stress and anxiety in stressed https://doi.org/10.3390/nu18020230
Nutrients2026,18, 230 7 of 22 adults, with reported effect sizes of Cohen’s d = 0.8–1.2 [56–58]. When converted to ANOVA metrics, this corresponds to an effect size of approximately f = 0.40–0.60. To avoid potentially underpowering the trial, the sample size calculation is conservatively based on a medium effect (f = 0.25). The analysis assumes an effect size of f = 0.25, a significance level ofα= 0.05 and power = 0.80, with a correlation of 0.5 among repeated measures. This calculation indicates that 34 participants are needed for statistical validity, with 17 in each of the ASH and PLA groups. The calculation was performed for the overall sample to ensure sufficient power for group-level effects, with results presented separately for men and women. A 2×2 mixed-design ANOVA was employed with Timepoints (Baseline, T1) as the within-subject factor and Condition (PLA, ASH)×Sex (male, female) as between-subjects factors. Variables showing a significant Condition×Time interaction were checked for baseline differences usingt-tests for normally distributed variables and Wilcoxon tests for non-normal variables. For outcomes with significant baseline differences, ANCOVA was applied using the baseline value as a covariate. Post hoc analyses were performed following the initial analyses and pairwise comparisons were adjusted using the Bonferroni correction to control for the increased risk of false positives, producing the finalp-value. Supplement satisfaction scores from both the ASH and PLA groups were analysed using Mann–Whitney U tests as scores were on a Likert scale, resulting in ordinal data. For each variable, a single 95% confidence interval (CI) was calculated for the combined sample (ASH and PLA) at each timepoint using the pooled mean and its standard error. The majority of variables deviated from normality, as indicated by Shapiro–Wilk tests. Data continued to be analysed with ANOVA, a statistical method that is robust to moderate deviations from normality when sample sizes are moderate and equal across groups (n= 14 per group/sex) [59]. 2.5. Processing of Saliva Samples Saliva samples were stored at−20 ◦ C until analysis. The samples were analysed by Dresden LABservice GmbH (Dresden, Germany). Alpha-amylase was measured using a Genesis RSP8/150 liquid handling system (Tecan, Germany).
Description
The study evaluates Ashwagandha's impact on stress and recovery in athletes.