**1. Introduction**

Maximum oxygen consumption (VO2max) is a physiological parameter that defines the aerobic capacity of a person. It is an indicator of the cardiorespiratory fitness that describes health status [1] and sport performance [2]. Focusing on competitive sports, the VO2max, together with running economy and the anaerobic threshold, is one of the main factors that determine success in endurance activities [3], and also contributes to increase the team sports performance by increasing work intensity, distance covered, and number of sprints completed [4]. However, from the point of view of the physical training, there are still controversies about the best training intensity to enhance the VO2max [5,6].

Apart from sport performance, VO2max has special interest in the field of health. Low values of VO2max (<17.5 mL·min-1·kg-1) are associated with an increased risk of mortality and loss of independent lifestyle in adults and elderly [7], while high values of cardiorespiratory fitness have been associated with a reduced risk of cardiovascular diseases [8,9]. The VO2max level is also important in children, where a higher aerobic capacity is related to better quality of life [10].

Ashwagandha (*Withania somnifera)* is a plant in the Solanaceae family. The extract of the Ashwagandha root has many biological implications due to its diverse phytochemicals [11], so it has been used, singly or in combination with other natural plants, in many research studies for its properties: anti-diabetic [12], anti-inflammatory [13], anti-microbial [14], anti-tumor [15], anti-stress [16], cardioprotective [17], or neuroprotective [18]. It also displays enhanced endothelial function [11], reduces reactive oxygen species [13], regulates apoptosis [19], and modulates mitochondrial function [11], showing to be effective to treat aging effects [20], anxiety and stress [21], arthritis [22], cognitive functions and memory [23], diabetes [12], epilepsy [24], fatigue [25], neurodegenerative diseases [26], pain [27], thyroid function [28], and skin diseases [29].

In spite of the relevant benefits of supplementation with Ashwagandha, only four meta-analyses have been carried out evaluating its efficacy on anti-inflammatory effects [30], on impotence and infertility treatment [31], on neurobehavioral disorders [32] and anxiety [33]. However, there are no meta-analyses that analyze the effect of Ashwagandha on physical performance. Therefore, the purpose of this study was to systematically review the scientific literature about the effects of supplementation with Ashwagandha on VO2max and to provide practical recommendations. Besides, a meta-analysis was carried out to determine the effect sizes of Ashwagandha on VO2max.

#### **2. Methods**

The review was conducted following the statements of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Guidelines (PRISMA).

#### *2.1. Literature Search*

To find the studies reported in the meta-analysis, several electronic databases were screened: PubMed (Medline), Web of Science (which includes other databases such as Current Contents Connect, Derwent Innovations Index, Korean Journal Database, Medline, Russian Science Citation Index, and Scielo Citation Index) and Google Scholar. The search was conducted in September 2019. The search terms were: (a) the type of treatment (Ashwagandha or "*withania somnifera*") and (b) the outcome variable ("oxygen consumption" or "aerobic" or "VO2"). The search was conducted using the treatment and the outcome variables, separated by the Boolean operator "and".

#### *2.2. Study Selection*

The inclusion criteria were: (a) VO2max data, with means ± standard deviation (SD) before and after the supplement intervention; (b) the study was a randomized controlled trial (RCT); (c) the article was written in English. Two independent authors selected the potentially eligible articles from the databases. There were no disagreements.

#### *2.3. Quality of the Evidence and Risk of Bias*

The quality of the evidence was categorized using the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) approach. The risk of bias was assessed by the Cochrane Collaboration's tool for assessing risk of bias. This tool classified the selection, performance, detection, attrition, and reporting bias into low, high, or unclear risk of bias.

#### *2.4. Data Collection*

Two authors independently extracted data from the studies. The information included: participants, interventions, comparisons, outcomes, and study design (PICOS), following the recommendations from the PRISMA statement. Table 1 shows age, sex, sample size, and condition of the participants. Table 2 presents intervention and the comparison groups, including type of supplementation with the doses, duration of the study, and the daily frequency of the supplementation. Figure 3 displays results for the different outcomes. Study design was not included in any table because all studies were RCT.


**Table 1.** Characteristics of the sample.

RCT: randomized controlled trial; AS: Ashwagandha group; M: males; F: females; CG: control group.


RCT: randomized controlled trial; KSM-66: commercial name of an Ashwagandha extract; WS: Withania Somnifera. Total dose was calculated as: total dose (g) = (dose (mg) × daily frequency × study duration (days))/1000.

## *2.5. Statistical Analysis*

The main outcome of this meta-analysis was VO2max. The meta-analysis was conducted using the Revision Manager (RevMan) software (version 5.3) obtained from Cochrane Collaboration web. Post-intervention mean and SD were extracted and used for meta-analyses. All articles reported VO2 max as mL/kg/min. Mean difference was calculated using a random model. The heterogeneity between the studies was calculated using Tau2, I2, and Chi2 tests. Although there is no consensus about the definition of "mild", "moderate", or "severe" heterogeneity, Higgins and Thompson [34] suggested that values for I<sup>2</sup> higher than 56% would mean large heterogeneity while values lower than 31% would be related to low heterogeneity.

#### **3. Results**

#### *3.1. Study Selection*

The PRISMA flow diagram is showed in Figure 1. A total of 129 records were identified, 9 of which were removed because they were duplicated. Of the remaining 120 articles, 92 were excluded because they were not related with the topic, 4 studies were not written in English, and 4 were reviews. After reading the remaining 20 articles, another 15 studies did not meet the inclusion criteria and were excluded. Therefore, 5 studies were included in the systematic review. However, the article by Sandhu et al. [35] was excluded from meta-analysis due to the odd results. In this regard, they evaluated healthy young males and females aged between 18 and 25 with body mass index between 18 and 25. Their mean peak VO2max was lower than 14mL/kg/min, which is so much lower than expected for healthy young people and less than half the mean of the rest of the included studies (46.18 mL/kg/min). We tried to contact with the authors in order to obtain a reason for that, but at the time this article was considered for publication, we did not receive a response. Considering that in the article authors did not explain an incremental test to obtain the VO2max, we believe that they measured the gas exchange at rest, reporting the oxygen consumption (VO2). Therefore, this article was included in systematic review but not in the meta-analysis.

**Figure 1.** Flow chart delineating the complete systematic review process.

## *3.2. Quality of Evidence and Risk of Bias*

The evidence of the effects on VO2max was initially classified as "high quality" due to all the selected articles were RCT, but the evidence dropped twice because of the small sample size and due to the high degree of heterogeneity. Therefore, the final quality of the evidence was low. The Cochrane Collaboration's tool for assessing risk of bias (Figure 2) showed that the poorer scores were obtained in the performance and detection bias due to unclear reporting.
