*3.4. Interventions*

The characteristics of the Ashwagandha supplementation and placebo group are displayed in Table 2. The doses varied from 300 to 500 mg and the daily frequency intake was once or twice a day. The total duration of the intervention varied from 2 to 12 weeks.

#### *3.5. Outcome Measures*

The study of Choudhary et al. [36] found a significant group\*treatment interaction in the VO2max. The remaining four articles only found within-group improvement in VO2max after the supplement intervention [35,37–39].

Regarding meta-analysis results, a significant (*p* = 0.04) mean difference was observed. Figure 3 showed a mean difference of 3.00 (95% CI from 0.18 to 5.82). The heterogeneity level was large according to the I2 = 84%. The quality of the evidence was low according to the GRADE classification.

*Nutrients* **2020**

, *12*, 1119

**Figure 3.** Meta-analysis results of the effects of Ashwagandha supplementation on VO2max.

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#### **4. Discussion**

The purpose of this study was to systematically review the scientific literature about the effects of supplementation with Ashwagandha on VO2max and to carry out a meta-analysis to determine the overall effect. After 20 articles were assessed for eligibility, 15 articles were excluded since they did not report VO2 max. A total of 5 articles were included in the systematic review [35–39]. However, one article was excluded from the meta-analysis [35] since the reported mean VO2max was abnormally low for healthy young people and less than half the mean of the rest of the included studies (46.18 mL/kg/min), which may indicate that they were not actually reporting VO2max but VO2 at rest. The results of this meta-analysis showed that supplementation with Ashwagandha may be useful to improve VO2max in athletes [36,38,39] and healthy adults [37]. Table 2 displayed the amount of Ashwagandha used in each study, which varied from 330 up to 1000 mg/day, which is inside the limits, 750 to 1250 mg/day, found to be well tolerated and safe [40]. In this regard, none of the five articles reported any relevant side effect as a consequence of the treatment, achieving a high compliance with the treatment and very low number of dropouts.

The two studies that achieved the highest treatment effect and effect size [36,39] were those with the highest Ashwagandha intake (>50 g in the whole program). Therefore, it seems like the higher the dose, the higher the improvement in VO2. However, the study by Tripathi, Shrivastava, Ahmad Mir, Kumar, Govil, Vahedi, and Bisen [14] did not observe any significant difference between the effects of a 330 mg intake and the effects of a 500 mg intake after 2 weeks. Therefore, further studies comparing the effect of different doses, as well as studies with longer duration are needed.

In general terms, the overall effects were better in those studies with a sample comprised of athletes [36,38,39] compared with the studies with healthy adults [14,39]. This is interesting since, as expected, baseline levels were higher in athletes and, consequently, larger improvements were expected in non-athlete healthy adults. It could be that the effects of supplementation with Ashwagandha might be linked to the physical activity levels of the participants, promoting and increasing the physiological adaptations to physical exercise. However, this hypothesis should be explored in future studies. The VO2max defines the body's ability to transport and utilize oxygen, so this physiological parameter is associated with endurance performance. Many factors contribute to the VO2max values, including genetic predisposition [41], enzymes [42], muscle fiber type [43], or training [44]. It is also known that nutritional supplementation can improve the effects of training and reach higher performance [45]. Previous studies with Ashwagandha administration observed improvement in working capacity test in rats by increasing the swimming endurance test [46]. As endurance performance is determined by mitochondrial function, some reasons for the Ashwagandha to improve cardiorespiratory fitness can be the significant effects observed on mitochondrial and energy levels, by reducing the succinate dehydrogenase enzyme activity in the mitochondria and benefiting Mg-ATPase activity [47]. Previous studies showed that Ashwagandha significantly enhanced the hemoglobin concentration and red blood cells in animals [48] and also in humans [38], with the subsequent increase in the capacity to transport oxygen to the muscles. Moreover, it should be considered that Ashwagandha has shown to have anti-fatigue [49,50] and anti-stress [51] actions. This could be connected to the significant improvement in the time to exhaustion of the experimental group that could be observed in the study of Shenoy, Chaskar, Sandhu, and Paadhi [39]. Some of the chemical constituents of *Whitania somnifera* [52] such as flavonoids, alkaloids, and steroidal lactones (withanolides) or the antioxidants (superoxide dismutase, catalase, and glutathione peroxidase) could be behind the improvements of VO2max. Therefore, further studies are needed to explore which are the chemical constituents and mechanism that may explain the potential improvement in the VO2max.

Although all mechanisms by which Ashwagandha can improve the VO2max have not been described yet and future studies are needed to elucidate that improvement, it is known that Ashwagandha exhibits little or no associated toxicity [53], so it seems that this Ayurvedic herb "Ashwagandha" (*Withania somnifera*) can be safely used for improving cardiovascular fitness in healthy adults and also in athletes, offering an additional alternative as a nutritional supplement to enhance VO2max.

Some limitations in the present meta-analysis can be mentioned. The first one is related to the search strategy, only articles published in English were included and a few databases were used. Another limitation can be the large heterogeneity in the included articles. Different doses, levels of physical activity, or the inclusion of both women and men in the protocols make it very difficult to achieve a high level of evidence. In addition, the systematic review and meta-analysis was not prospectively registered in any public database. Furthermore, in order to have a better understanding of long-term ergogenic benefit and potential side effects from Ashwagandha root extract, longer duration studies are needed.
