This randomized, double-blind, placebo-controlled study was conducted at an independent research organization according to the Declaration of Helsinki in agreement with the International Conference on Harmonization guidelines on Good Clinical Practice. The study protocol was approved by the Institutional Ethics Committee (IEC) of the Vydehi Institute of Medical Sciences and Research Center (Bengaluru, Karnataka, India) and registered in the Clinical Trials Registry (CTRI) of India (Registration no. CTRI/2018/12/016641).

Healthy men, 19–29 years, with a body mass index (BMI) of 18.5–29.9 kg/m², with a normal electrocardiogram, and who were previously untrained in resistance or power exercise for at least 6 months, were recruited for the study. Men with a history of alcohol or drug abuse, sleep disorder, eating disorder, or psychiatric condition, or those needing to fast as a personal practice, were excluded from the study. Additional exclusions included men who used protein or had taken any dietary supplements containing protein, creatine, HMB, or other supplements thought to improve muscle strength and/or muscle mass, or used corticosteroids, testosterone, or anabolic drugs in the three months before the study initiation. Each volunteer was aware of the study protocol and provided written informed consent before any study-related procedures were conducted.

A total of 120 subjects were enrolled and randomized into one of four equal groups (n = 30) by following the randomization codes as generated by SAS procedure PROC PLAN using block design: (A1) 425 mg SMI plus one RET set; (A2) 850 mg SMI plus one RET set; (P1) placebo plus one RET set; and (P2) placebo plus two RET sets. This study involved five scheduled visits, screening (days −14 to −1), randomization/baseline (day 1), and days 14, 28, and 56. The study products were labeled by randomization code and dispensed to the participants on study days 1, 14, and 28. Subjects were instructed to consume two capsules every morning for 56 days. Compliance with the supplementation regimen was monitored through daily diary recordings of capsule consumption and the collection/counting of all unused capsules. All subjects were provided a training regimen as per their group assignment and compliance with the training regimen was calculated as the total number of sessions completed under the supervision of trained instructors and study personnel per week for 8 weeks. Subjects were instructed to maintain their habitual dietary intake and physical activity levels, except for their assigned RET program, during the study. Participants completed written 3-day diet records for two weekdays and one weekend day at pretraining and in the last week of the training period and these diet records were evaluated for consistent eating patterns. Adverse events, tolerance, and vital signs—heart rate, blood pressure, temperature, respiratory rate—were assessed throughout the study and recorded at each study visit. Body weights were measured with a standard scale in triplicate to the nearest 0.1 kg and the average was recorded at each visit. Fasting blood samples were collected at screening, baseline, and day 56. Performance testing occurred at baseline and on days 14, 28, and 56. All participants, investigators, and study personnel remained blinded to treatment assignment for the duration of the study.

The botanical supplement, SMI, is a water-dispersible version of a formulation containing a blend of extracts from S. indicus flower heads and M. indica L. stem bark at 50.03% (w/w) herbal blend and 49.97% (w/w) excipients. The excipients include dewaxed shellac (33.3%), guar gum (8.33%), citric acid (6.67%), and colloidal silicon dioxide (1.67%). The final product was standardized to contain not less than 2.5% 7-hydroxy frullanolide and 2% mangiferin following methods of preparation and analytical procedures as previously described for the non-water dispersible formulation (20). The investigational products and placebo were manufactured under a strict Good Manufacturing Process, packaged, and labeled by randomization code by Laila Nutraceuticals (Vijayawada, India). SMI is commercially available as Myotor^® and RipFACTOR™ from PLT Health Solutions (Morristown, NJ, USA).

The two active groups (A1, A2) and one placebo group (P1) completed the same exercise protocol, whereas the second placebo group (P2) completed double the number of RET sets. A second placebo group completing double the number of RET sets was deployed as a type of positive control, as research supports that an increased volume of weighted-load training can increase strength gains in a dose-response relationship (1, 3). Given the potential for uncaptured differences in individual work efforts within our cohort of untrained subjects, we leveraged this four-group experimental design with two exercise-dose comparator groups to help ensure any measured strength differences between treatment and placebo could be attributed to treatment effects.

All the volunteers completed RET on three non-consecutive days per week for 8 weeks under the supervision of a certified trainer and spotter. At each of the 24 training sessions, the subjects appropriately warmed up and then completed one set (for groups A1, A2, and P1) or two sets (group P2) of 6–8 repetitions of both bench press (BP) and leg extension (LE), at 80% of their most recent individualized muscle strength score (1-RM) assessment. The 1-RM, determined for each subject as the heaviest lift completed within a series of single repetitions of progressively heavier weights until failure, was assessed at baseline, day 14, and day 28. The number of repetitions and sets remained constant at each training session as per the assigned group.

The primary endpoint was the change in muscle strength, as measured by 1-RM in BP and LE exercises, from baseline to the end of the study. Each subject’s 1-RM for BP and LE was summed to obtain a measure of total muscle strength. Secondary endpoints were changes in muscle endurance, measured as the number of repetitions lifted at 80% of the subject’s baseline 1-RM until failure, and changes in serum concentrations of FT, TT, and C from baseline to day 56.

Safety parameters, including complete blood cell counts and blood chemistry— liver function tests, renal function tests, plasma total cholesterol, low- and high-density lipoprotein (LDL, HDL) cholesterol, triglycerides, and fasting glucose—were assessed from fasting blood samples taken at screening and day 56. Test parameters for urine analysis included specific gravity, pH, albumin, bile salts, bile pigment, glucose, red blood cells, and ketones.

Muscle strength was determined by 1-RM measurements for BP (Olympic Flat Bench) and LE (StayFit Single Station) on days 0, 14, 28, and 56. Strength assessments followed the American Society of Exercise Physiologists’ procedure for accurate assessment of muscular strength (22) and were done under the supervision of a certified athletic trainer and spotters. Standardized instructions were provided on lifting techniques and testing procedures, standardized weights and bars were utilized, and verbal encouragement was provided during testing. After an appropriate whole-body warm-up, subjects performed two practice sets of 6–8 repetitions at approximately 60 and 80% of their estimated 1-RM. Subsequent lifts were single repetitions of progressively heavier weights until failure. The 1-RM was determined after three to five attempts at the heaviest single repetition weight, with a rest interval of 2–4 min between attempts.

Muscle endurance was measured as repetitions to failure (RTF). RTF was determined by measuring the maximal number of repetitions a subject completed when lifting 80% of the established baseline 1-RM until failure. To assess a fixed measure of relative muscular endurance, the original baseline 1-RM was always utilized for muscular endurance testing throughout the study.

Serum hormones were measured from fasting blood samples collected by direct vein puncture using standard enzyme immunoassay (ELISA) kits. The assay procedures for measuring TT, FT, (DRG International, Inc. Springfield, NJ; TT intra-assay coefficient of variation [CV] 3.3–4.2%, inter-assay CV 4.7–9.9%, sensitivity 0.083 ng/ml; FT intra-assay CV < 10%, inter-assay CV < 10%, sensitivity 0.06 pg/mL), and C (Cal Biotech Inc., El Cajon, CA; sensitivity 20 ng/mL) followed the protocols provided by the vendor. Each serum sample was tested in duplicate. The ELISAs were based on the principles of sandwich immuno-enzymatic reactions. The supplied substrate solutions developed the color reactions, and a microplate reader (Bio-Rad Laboratories, Hercules, CA) recorded the absorbance. The analyte concentrations in the serum samples were calculated from the standard curves plotted in each assay. The assay variation was calculated across 4 plates of 5 (FT) or 6 (TT and C) standards, and the intra- and inter-assay CVs were 0.7 ± 0.4% and 4.7 ± 0.5% for FT; 1.3 ± 0.6% and 3.8 ± 0.4% for TT; and 1.5 ± 0.9% and 10.7 ± 1.9% for C.

Urine, serum, and whole blood parameters were assessed in all participants at the baseline and final visits. Biochemical parameters were measured using Cobas C 311 (Roche Diagnostics, Rotkreuz, Switzerland), and hematological parameters were measured using the auto hematology analyzer Mindray BC-20 (Shenzhen Mindray Bio-Medical Electronics Co., Ltd, China). Urine analysis was carried out using standard Siemens Multistix 10 SG reagent strips and by microscopy of sediment.

The data were analyzed using SPSS Software version 29.0 (SPSS, Inc., Chicago, IL, USA). The normality of the data was assessed using the Shapiro-Wilk test. Where assumptions were met, data were analyzed by a 4 × 3 mixed factorial group (A1, A2, P1, P2) by time (14, 28, 56 days) ANCOVA or a univariate ANOVA (day 56) with the baseline measurement held as a covariate. Main effects (time or group) and interaction (time × group) effects were evaluated after the degrees of freedom were corrected by Greenhouse-Geisser for sphericity. The calculation of partial effect size (η²) was used to clarify the magnitude of the main effects, defined as small, 0.01; medium, 0.06; and large, 0.14. When the F-ratio was significant for main or interaction effects, a priori planned pairwise comparisons at each time point were conducted using paired (intragroup) or unpaired (intergroup) Student t-tests adjusted for multiple comparisons using the Bonferroni correction. Data not meeting normality assumptions were analyzed with non-parametric methods: Friedman repeated measure ANOVA by rank for within-group effects and Kruskal–Wallis ANOVA to compare groups at the same time point, adjusted by Bonferroni correction for multiple tests. Effects were considered significant at p < 0.05. General characteristics and descriptive statistics are expressed as mean ± standard deviation (SD), whereas the estimated marginal means and calculated mean differences (MD) are presented as means ± standard error of the measurement (SE) and reported in conjunction with the 95% confidence interval (CI).

A priori analysis utilizing pilot data determined that a sample size of at least 18 subjects per group would be required to achieve 80% power to detect a significant difference at 0.05 alpha level. With an estimated dropout rate of 20–30%, recruitment was determined at 120 subjects. One hundred and sixty-five participants were assessed for eligibility, 13 failed screening, and 32 voluntarily withdrew. After 120 subjects were randomized into 4 equal groups (n = 30), eighteen subjects dropped out due to scheduling difficulties before supplementation began, one dropped out during the training phase because of a non-study-related adverse event, and two subjects were removed due to protocol deviations. In total, 99 subjects completed the study: A1, n = 26; A2, n = 25; P1, n = 25; and P2; n = 23. The flow of the study progress is shown in the consort diagram (Figure 1).

The four groups were comparable in age, anthropometric data, and baseline measures of muscle strength, endurance, and hormones before the treatment began (Table 1). There were no serious adverse events reported during the study. Eight subjects experienced minor adverse events which were evenly divided between placebo and supplement. Specific minor adverse events in the supplemented groups included two subjects with superficial limb pain/abrasions due to minor incidents occurring outside the study, and two with generalized body aches, headache, fever, and abdominal discomfort. Adverse events within the placebo groups consisted of one subject with minor symptoms resulting from a scorpion sting, one with complaints of itching, and two with generalized body pain, fever, headache, and chills. These adverse events were self-limiting and resolved fully during the study. All subjects completed 100% of their assigned training sessions and daily consumption of study products was > 90%. Written diet records indicated dietary patterns did not alter from the beginning to the end of the study, and body weights were stable or slightly increased (Table 2). Safety parameters and vital signs were within normal clinical ranges at baseline and the end of the study (Supplementary Tables 1, 2).

Analysis of BP strength as measured by 1-RM is presented in Tables 2, 3. The analysis demonstrated a significant main effect of time (p = 0.008, η² = 0.064), group (p < 0.001, η² = 0.202), and time × group interaction (p < 0.001, η² = 0.181). All groups significantly (p < 0.05) improved BP strength from their baseline measures (Table 2), but supplementation improved strength compared to the exercise-only groups. Compared to P1, A1 lifted 3.3 ± 1.1 kg (95% CI 0.5–6.2, p = 0.012) more on day 14, 5.7 ± 1.8 kg (95% CI 0.7–10.6, p = 0.016) more on day 28, and 10.6 ± 3.0 kg (95% CI 2.6–18.6, p = 0.003) more on day 56 (Table 3). A2 increased BP strength compared to both P1 and P2. A2 lifted 6.5 ± 1.9 kg (95% CI 1.5–11.5, p = 0.004) more on day 28, and 13.8 ± 3.0 kg (95% CI 5.7–21.8, p < 0.001) more on day 56 than P1 (Table 3). A2 bench pressed 5.5 ± 1.9 kg (95% CI 4.1–10.6, p = 0.027) more on day 28 and 9.9 ± 3.1 kg (95% CI 1.7–18.2, p = 0.010) more on day 56 than P2 (Table 3). The difference in BP strength between A1 and A2 was not significant at any time point. Likewise, P1 and P2 were not statistically different at any time point.

Analysis of LE strength demonstrated a significant main effect of time (p < 0.001, η² = 0.264), group (p < 0.001, η² = 0.245), and time × group interaction (p = 0.007, η² = 0.100). Measured values and pairwise group comparisons are shown in Tables 2, 3. All groups significantly (p < 0.05) improved over time in LE strength from their baseline values (Table 2), but the supplemented groups lifted more weight than the exercise-only groups. A1’s LE strength measures were greater than that of P1 at each time point, with A1 lifting 5.9 ± 1.6 kg (95% CI 1.5–10.3, p = 0.003) more on day 14, 8.3 ± 1.9 kg (95% CI 3.2–13.3, p < 0.001) more on day 28, and 8.2 ± 2.2 kg (95% CI 2.4–14.0, p < 0.001) more on day 56 (Table 3). A1 also improved more than P2 (p < 0.05) on days 28 and 56 (Table 3). A2, the group supplemented with the higher amount of SMI, demonstrated significantly (p < 0.05) more strength compared to P1 at all time points and compared to the placebo group assigned to double RET sets, P2, on days 28 and 56 (mean difference of 6.9 ± 1.9 kg, 95% CI 1.6–12.1, p = 0.004 and 7.9 ± 2.2 kg, 95% CI 1.9–13.9, p = 0.003, respectively; Table 3). Measures of LE strength were not statistically different between A1 and A2, nor between P1 and P2 at any time point.

There was a significant main effect of time (p < 0.001, η² = 0.129), group (p < 0.001, η² = 0.322), and time × group interaction (p < 0.001, η² = 0.217) for total muscle strength. Measured values and pairwise group comparisons are illustrated in Tables 2, 3. Within-group comparisons illustrated that all groups significantly improved over their baseline values in total muscle strength (Table 2). Both supplemented groups, A1 and A2, improved their total muscle strength compared to P1 (p < 0.001) at all measured time points (Table 3). A1 improved more than P2 on days 28 (p = 0.003) and 56 (p = 0.018). A2 also improved (p < 0.001, Table 3) total muscle strength compared to P2 on day 28 (mean difference of 12.3 ± 3.0 kg, 95% CI 4.2–20.4) and day 56 (17.7 ± 4.1 kg, 95% CI 6.6–28.9). Total muscle strength between A1 and A2 and between P1 and P2 were not statistically different at any time point.

All groups demonstrated improved muscular endurance as the number of BP-RTF and LE-RTF increased over time (p < 0.001 for each group, Table 4). Non-parametric Kruskal–Wallis analyzed at each time point illustrated a significant group effect for BP-RTF on day 28 (p = 0.040) and day 56 (p = 0.003, Table 4). A2 significantly (p < 0.05) improved BP-RTF compared to P1 on days 28 and 56 and compared to P2 on day 56 (Table 4). For LE-RTF, a treatment effect trended toward significance on day 28 (p = 0.071) but only reached significance (p = 0.005) on day 56. Pairwise comparisons illustrated that both supplemented groups, A1 and A2, improved LE-RTF compared to P1 (p < 0.05) but not P2 at day 56 (Table 4).

The baseline and day 56 values of serum hormones are presented in Table 5. Statistical analysis revealed a significant main effect of time (p < 0.001, η² = 0.292) and a significant time × group interaction effect (p = 0.016, η² = 0.103) for FT values. There was also a significant time (p < 0.001, η² = 0.193) and time × group interaction effect evident for serum TT (p = 0.017, η² = 0.102). Both supplemented groups (A1, A2) but neither of the exercise-only placebo groups (P1, P2) significantly (p < 0.05) increased mean FT and TT values from baseline to day 56 (Table 5). The final FT and TT values for A1 and A2 were significantly (p < 0.05) elevated compared to those of P1, but not compared to P2 (Table 5).

Analysis of mean C values demonstrated a significant time × group interaction effect (p = 0.034, η² = 0.087; Table 5). There was not a significant main effect of time nor were there significant changes from baseline values in any group. However, the analysis revealed a significant (p < 0.05) difference between A2 and P2, as A2 decreased from measured baseline values while P2 increased.