The Impact of 12-week Ashwagandha Supplementation Induced by Resistance Training on Strength, Levels of Neurotrophic and Stress Response Proteins, Oxidative and Psychological Stress, and Cognitive Function in Physically Active Male Adults

This randomized, double-blind, placebo-controlled clinical trial aims to investigate the effects of 12-week supplementation with Ashwagandha (Withania somnifera) extract on a range of physiological, biochemical and psychological outcomes in healthy adult males, experienced in resistance training.

Ashwagandha is an adaptogenic herb commonly used to support stress resilience, sleep quality, hormonal balance and physical performance. Although prior studies suggest promising benefits in these areas, current evidence is limited in scope and often lacks methodological rigor. Specifically, there is a scarcity of research examining Ashwagandha's effects in physically active individuals and in combination with structured exercise programs.

The goal of this study is to assess whether Ashwagandha supplementation enhances physiological adaptations to resistance training and/or improves health markers in trained individuals. Researchers will also examine the underlying mechanisms by evaluating hormone levels, neurotrophic factors, oxidative stress, inflammation and their relationship with performance and well-being outcomes.

A total of 40 healthy male participants will be recruited and randomly assigned to one of two groups:

• Resistance-trained individuals + resistance training + Ashwagandha (n = 20)
• Resistance-trained individuals + resistance training + placebo (n = 20)

All participants will undergo pre- and post-intervention assessments. Participants will follow a structured resistance training program consisting of three weekly sessions for 12 weeks. Those in the intervention group will receive 400 mg/day of Ashwagandha extract (KSM-66, standardized to 5% withanolides), while control group will receive visually identical placebo capsules.

Primary outcome measures include:

Hormonal and biochemical markers: cortisol, testosterone, IGF-1 - Insulin-like Growth Factor 1, BDNF, and oxidative stress markers (e.g., lipid and protein peroxidation).

Neurocognitive and psychological assessments: cognitive performance (VPT, VFT - Verbal Fluency Test, Stroop Test, TMT-A - Trail Making Test, Part A, TMT-B - Trail Making Test, Part B), perceived stress (PSS-10 - Perceived Stress Scale), and sleep quality (PSQI - Pittsburgh Sleep Quality Index), mood (POMS - Profile of Mood States).

Physical performance: maximal strength (one repetition maximum, 1RM bench press, Biodex), muscular endurance (bench press repetitions to failure at 75% 1RM).

Body composition: skeletal muscle mass and body fat percentage measured via bioelectrical impedance (Seca mBCA 555).

Participants will be monitored for compliance via supplement counts and electronic logs. Dietary intake will be tracked weekly using structured food diaries and analyzed with the Fitatu application. One training session a week will be supervised and logged by certified strength and conditioning coaches, another two will be performed by participants themselves and monitored through an electronic log.

Blood samples will be collected at baseline and post-intervention for hormonal, inflammatory, and oxidative stress analysis. Serum and plasma will be stored at -80°C and analyzed using ELISA and spectrophotometry methods.

The study's main hypotheses are as follows:

Ashwagandha supplementation combined with resistance training will improve hormone profiles (increase testosterone, IGF-1, BDNF; decrease cortisol, stress markers) more than placebo conditions.

Ashwagandha will enhance physical performance (increase maximal strength, muscular endurance, skeletal muscle mass; decrease body fat) beyond that of placebo conditions.

Ashwagandha will reduce perceived stress and improve sleep quality and cognitive function.

Positive correlations are expected between improvements in biomarkers (e.g., BDNF, testosterone) and performance and psychological outcomes.

This study addresses several gaps in the current literature by combining objective physiological and psychological measures, investigating potential mechanisms of action such as oxidative stress and inflammation. Unlike previous research, this trial will examine integrated outcomes rather than isolated variables, allowing for a more comprehensive understanding of Ashwagandha's effects.

Participants will be recruited via social media campaigns and corporate outreach events. Safety monitoring will include weekly contact, adherence checks, and documentation of adverse events. Ashwagandha's safety profile is supported by prior studies using comparable doses (400-600 mg/day), which report low incidence of side effects. Nonetheless, participants may withdraw at any time and will be excluded in the event of injury, illness, or withdrawal of consent.

Ethical approval has been granted, and the study is conducted in accordance with the Declaration of Helsinki. The results are expected to contribute to evidence-based recommendations for adaptogenic supplementation in both trained and general populations. The research team includes experts in physiology, sports nutrition, neuropsychology and clinical supplementation research, ensuring a rigorous and interdisciplinary approach.

By examining how Ashwagandha interacts with physical training and how it affects biological, cognitive and psychological outcomes, this project will help define the contexts in which Ashwagandha can be most effectively used. The study's findings may also have implications for stress management, cognitive health, and performance enhancement strategies in health-conscious or physically active populations.

Inclusion criteria

• Minimum 6 months of consistent resistance training experience
• Participation in at least two resistance training sessions per week
• No use of Ashwagandha, adaptogenic supplements, or medications
• No medical contraindications and signed informed consent

Exclusion criteria

• History of cardiovascular events, liver disorders, or psychiatric illness
• Medical contraindications for participation
• Use of nicotine products, including e-cigarettes
• BMI > 29.9, to ensure homogeneity in body composition