Effects of Ashwagandha (Withania somnifera) standardized root extract on physical endurance and VO_2max in healthy adults performing resistance training: An eight-week, prospective, randomized, double-blind, placebo-controlled study

Ethical approval

The study was approved by the Institutional Ethics Committees of both King George Medical University (Reference number. #202/IEC/R.Cell-18) and M. V. Hospital and Research (Reference number #IEC/01/28/18) and it was prospectively registered with the Clinical Trials Registry of India (Registration# CTRI/2018/07/014969).

Study design

This was a parallel-group=eight-week, multicenter, randomized, double-blind, PB-controlled study. It was conducted in compliance with the Declaration of Helsinki guidelines, the New Drugs and Clinical trials rules 2019 and the Indian Council of Medical Research (ICMR) ethical guidelines for biomedical research on human subjects.

Recruitment and randomization

The recruitment of participants was performed by circulating printed fliers in the purlieu of a gymnasium which served as the site for the training program. This brochure was approved by both institutional ethics committees. Recruitment of participants started on 11 September, 2018 (first visit of first participant) and ended on 28 March, 2019 (last visit of last participant). Subjects were randomly and equally allocated to two groups in 1:1 ratio using stratified randomization (male and female) to receive either an AG capsule standardized root extract (n=40), or PB which looked identical to AG capsule (n=40). The randomization was computer-generated through randomly permuted blocks of 20. Within each block, the number of participants assigned to each of the two treatment arms was equal. The AG and PB capsules were manufactured and packed in identical containers and labeled equivalently, along with unique serial numbers to ensure blinding. The study centers received numbered and sealed envelopes that contained no information about treatment allocation.

Subjects

All participants read and signed an informed consent form before final selection and enrollment in this study. Healthy adults of either sex aged between 18 to 45 years engaging in regular physical activity (gymnasium/strength training exercise at least three months before screening for this study) were considered eligible. Sex of the participants was defined based on self-reporting. Personal history and habits were found similar in both AG and PB groups. Participants agreed to engage in the same exercise schedule and diet regime for the study period as prescribed in the study protocol and if of child-bearing age, agreed to use barrier birth control measures. Participants were excluded if they were taking any nutritional supplements, medications, or steroids used to enhance physical performance. Those with a history of drug abuse, who smoked more than 10 cigarettes a day, or were habitually consuming more than 14 grams of alcohol per day were also excluded. Other exclusion criteria were: any planned participation in any sporting event during the study period; a weight loss of >5 kg in the previous three months; a history of any orthopedic injury or surgery in the past six months; any known hypersensitivity to AG; participation in any clinical study in the past three months; a history of heart disease, diabetes, depression, stroke or neurological disorder. Participants were also excluded if they were taking anti-hypertensive drugs, beta-blockers, beta-agonists, hormonal contraceptives, corticosteroids, or psychotropic substances over the previous three months.

Interventions

Capsules containing either 300 mg of AG standardized root extract (KSM-66, Ixoreal Biomed, CA, US) or 300 mg starch (Shri Kartikeya Pharma, Hyderabad, India) were used for AG and PB groups respectively. Both the AG and PB formulations were cellulose-based hard capsules and identical in appearance, weight, texture and color. Participants were required to take one capsule twice daily with milk or water. Two males and one female consumed capsules with milk in AG group, whereas 3 males and one female in PB group consumed capsules with milk.

The AG root extract is a light yellowish powder extracted using a proprietary process. The green extraction process is devoid of any alcohol or similar solvents, hence maintaining the standards as recommended by traditional Ayurveda. The product, KSM-66 Ashwagandha, contains a high concentration of root extract of this herb and more than 5% of total withanolides as standardized by high-performance liquid chromatography. The batch number of product used was KSM/VG/18/1020. The chemical profile of the study product was confirmed by Shri Kartikeya Pharma, Hyderabad, India.

Sample size

The target sample size was estimated using G*Power (Version 3.1.9.3). It was based on a previously published randomized controlled clinical study evaluating the effect of an AG root extract on muscle strength and recovery.¹⁸ A recently published systematic review on AG also supported this kind of improvement (effect size of 0.67) in physical performance.¹⁹ Considering the previous study, we hypothesized that AG treatment is better than PB by an effect size of 0.6 with regards to change in the one-repetition maximum (1 RM) bench press exercise after eight weeks. To detect an effect size of 0.6 in a two-parallel-group design (1:1) using independent Student t-test, with a 5% risk of type 1 error (alpha) and 80% power, 40 subjects per group were required while considering a 10% drop-out rate. Thus, 80 healthy male and female subjects were recruited in the study.

Resistance training exercise program

The resistance training regimen used in this study was focused on improving muscle strength, increasing muscle size and enhancing cardiorespiratory endurance. Various resistance exercises were chosen with the objective of training targeted muscle groups. Such training programs target the upper and lower body and consist of a series of exercises with suitable resistance, grouped into multiple sets and repetitions as per the National Strength and Conditioning Association’s (NSCA) regulations and guidelines.²⁰ Each participant was required to complete the training session every alternate day with a one-day complete rest per week. Therefore, participants were training three days per week. Each training session started with a warm-up of low intensity aerobic exercise. Participants were instructed to perform the maximum repetitions possible for each set until exhaustion. The exercise program is detailed in Figure 1, comprising exercises for week 1, week 2 and remainder of the study (weeks 3 to 8).

Figure 1. Exercise protocol.

Data for this study is available in the online repository ‘Figshare’ under the dataset named ‘Ashwagandha_Muscle study’ (Underlying data).³⁴

Study outcomes

Primary outcome measures

Muscle strength

Changes in upper and lower body muscular strength were the primary efficacy assessment of this study. As per common practice in sports medicine, muscle strength was assessed by one-repetition maximum (1RM) for bench press and leg press. 1RM testing was performed using NSCA’s protocol.²⁰ 1 RM refers to the maximal load lifted by a participant for one cycle of the exercise. Muscle strength measurements were conducted at baseline (first day of training) and end of the study (after the completion of eight weeks of training and supplementation). The equipment used was a Bench Press (SL7028) manufactured by Impulse and a Leg extension (GCEC340) manufactured by Body Solid Inc.

Secondary outcome measures

Muscle girth

Muscle size was measured at three sites - arm (flexed mid-upper arm), chest (sternum at mid-tidal volume) and upper thigh (just inferior to gluteal fold) of each participant. Measurements were undertaken on the first day of training (baseline) and at end of the study (day 56). Maximal cross-sectional area (CSA) was measured for thigh and arm using the Moritani-DeVries method,²¹ whereas, for chest measurement, girth was measured at the level of middle of the sternum by passing a measuring tape under both arms at the end of normal expiration.

Cardiorespiratory endurance (maximum rate of oxygen consumption)

Resistance training and endurance are highly correlated.^22,23 Cardiorespiratory endurance measures overall body performance during high-intensity exercises. Studies indicate that resistance training induces an increase in the maximal rate of oxygen consumption (VO_2max).^22,23 VO_2max is measured during incremental exercise, most typically on a motorized treadmill. Bruce protocol was used to assess the treadmill test outcomes of all participants. Test score was considered as time taken for the test in minutes and it was converted to estimate a VO_2max score as per the Bruce protocol.²²

Safety assessment and adverse events

Clinical safety was assessed based on the frequency of adverse events reported by the participants. In addition to this subjective report, standard biochemistry tests (hematology, renal function, liver function and thyroid function tests) were also performed along with measurement of vital signs.

Statistical analysis

Data was entered in Microsoft Excel spreadsheet using manual double-entry method to ensure data accuracy. Statistical analyses were done using MedCalc (version 20.011). Efficacy analysis was done on the modified intention to treat (ITT) dataset (n=73), and safety analysis was done on the whole dataset (n=80). Data for continuous variables are presented as means with standard deviation (SD), whereas categorical and discrete data are presented as counts with percentages. Unpaired t-test was used to assess between-group differences. Change in values form baseline to eight weeks were computed from and compared between the two groups using unpaired t-test. Effects of sex, BMI and chest circumference on different parameters were analyzed using analysis of covariance (ANCOVA). Adjusted means with 95% confidence intervals (CI) and effect size (Cohen’s d) were presented for change from baseline. Criteria for effect sizes were based on the standard criteria (<0.2, trivial; 0.2–0.6, small; 0.6–1.2, moderate; 1.2–2.0, large; 2.0–4.0, very large; and >4.0, nearly perfect).²⁴

Normality assumptions were checked on all variables using a one-sample Shapiro-Wilk test. A p-value lower than 0.05 was considered the threshold to claim statistical significance.

Participant demographics

A total of 92 people were screened for participation in the study, of which 80 met the inclusion criteria and were enrolled in the study (Figure 2). All enrolled participants completed the eight-week follow-up. However, four subjects (3 males and 1 female) in the PB and three subjects (3 males) in the AG group did not consume supplementation after their second visit. These participants were documented as having poor compliance to treatment and were excluded from the efficacy analysis. Reasons for premature medication discontinuation were reported as unintentional forgetfulness due to travel and other personal issues by these seven subjects. Baseline demographic characteristics and vital signs are detailed in Table 1 and indicate that the study population was homogenous with no significant differences between the treatment and control groups. Baseline parameters across both the groups are displayed in Table 2.

Figure 2. CONSORT flow chart.

Muscle strength

Changes in 1 RM bench press and leg press across both treatment groups throughout the eight-week trial period are detailed in Table 3. There were significant differences between the groups in both 1 RM bench press (males: effect size, 0.91; p=0.0084; females: effect size, 1.56; p=0.0005; total: effect size, 1.14; p<0.0001) and 1 RM Leg press (males: effect size, 1.22; p=0.0049; females: effect size, 0.63; p=0.018; total: effect size, 1.11; p=0.0005). A within-group analysis in AG group demonstrated a significant (p<0.0001) increase of 23.5 % and 22.8% in 1 RM bench press and an increase of 15% and 9.9% in 1 RM leg press over time and in both male and female participants respectively. Table 4 presents the comparison of AG and PB for change (unadjusted and adjusted for baseline values) in 1 RM leg press and 1 RM bench press from baseline to eight weeks.

Muscle size

Changes in muscle size across the two treatment groups during the study period are detailed in Table 3. At the end of the study, there was a significant increase in both chest circumference (Total participants: p=0.019) and arm circumference (Total participants: p<0.0001), among the participants in AG group as compared to the PB group. However, no improvement was seen in thigh circumference when compared to PB group. A within-group analysis in AG group demonstrated a significant increase in the chest circumference (males: p<0.0001; females: p<0.0001), mid-arm circumference (males: p<0.0001; females: p<0.0001) and thigh circumference (males: p=0.0003; females: p<0.0001). Table 4 presents the comparison of AG and PB for change (unadjusted and adjusted for baseline values) for mid-arm, thigh and chest circumference from baseline to eight weeks.

Endurance

Changes in the VO_2max across the two treatment groups throughout the eight-week trial period are detailed in Table 3. At the end of the study, there was a statistically significant between-group difference in VO_2max values (males: p<0.0001; females: p=0.0001), when compared with the PB group. In the AG group=a statistically significant 9.5% and 7% increase were observed over time in both males (p<0.0001) and females (p<0.0001), respectively. Table 4 presents the comparison of AG and PB for change (unadjusted and adjusted for baseline values) in VO_2max from baseline to eight weeks. The analysis for VO_2max yielded 100% power and greater changes were observed with AG as compared to placebo (effect size, 1.034, p<0.0001).

Adverse events and safety parameters

The participants did not report any adverse events during the study period. No significant changes were observed from baseline to week 8 in any of the laboratory tests conducted for hematology, renal function, liver function and thyroid function tests with both AG and PP treated participants (supplementary data). Similarly, no changes in any of the vital parameters was observed at week 8 in both treatment groups.³⁴