This was a randomized controlled trial of trained healthy Indian males taking part in the BRJ intervention study. The study protocol was approved by the ethical committee of the institute (Defence Institute of Physiology and Allied Sciences, Delhi). All the participants were informed about the study and read and signed the informed consent forms. They were instructed to refrain from consuming alcohol and smoking throughout the trail. A total of 126 individuals were recruited, Control (n=63), BRJ supplemented (n=63). All the participants included in this study were healthy, similar food (dining from the same mess and ad-libitum) and engaged in routine physical activity, none of the participants from both the groups had a previous exposure to high altitude. Exclusion criteria involve: Individuals with history, sign, symptoms or medication of any chronic disorders like diabetes, cardiovascular disease or neurological disorder.

SL measurements were taken at an altitude of 122m. After SL measurements, all the participants were randomized into two groups (Group I-Control and Group II-BRJ supplemented) and each group comprised 63 individuals. All the participants were inducted to HA (Western Himalayas-3300m) via road (it took about 12hrs to attain the desired altitude). Thirty-four participants have been dropped out of the study due to unavoidable circumstances (logistic problems) and a total of 92 participants have taken part in the study (Figure 1).

Beetroot supplementation was provided in the form of beetroot powder packed in individual sachets (spray dried-40gms), reconstituted in 200ml of water before consumption for 15 days. The Control group remained devoid of any supplementation during this period. All participants from the test group consumed this juice every day in the morning after breakfast. All the measurements were repeated on the 3^rd day (HA0) of arrival at HA before starting supplementation and on the 8^th (HA1) and 16^th day (HA2) of supplementation.

All the participants of the study completed the self-reporting Lake Louise AMS questionnaire every day in the morning for seven consecutive days after reaching HA. They scored their symptoms (headache, gastrointestinal symptoms, fatigue, and dizziness) on a scale of zero to three (0-no symptom, 1-mild, 2-moderate, and 3-severe). The overall AMS score is the sum of all the values from each category. An individual will be diagnosed with AMS if had a Lake Louise Score (LLS) of ≥ 3 15.

All the measurements and sample collection were done in the morning (6-8 am) including height and weight. Each participant rested for 10-15 min in a supine position before the measurement of SBP and DBP using a mercurial BP apparatus (Diamond Deluxe, India) along with HR and SpO₂ using finger pulse oximeter (Nonin Medical Inc. USA). Whole blood was collected from the antecubital vein after fasting of 12 hours in EDTA vacutainers and used to measure hematological parameters (H 360, Erba, Slovakia), within 30 minutes of collection.

Body composition was measured using Bioscan 920-II, a multi-frequency analyzer (Maltron, UK). All participants were restricted from wearing any metal on their bodies during the test. Each of them was instructed to be in a supine position with arms and legs apart. The electrodes were firmly attached to the carpal, meta-carpal, talus, and, metatarsal. The test was performed using the right side of the subject. The test was performed at each time interval except HA2 due to some logistic issues.

Queen’s College Step Test 16 is a submaximal exercise test, used to evaluate the maximum oxygen consumption (VO₂ max). The test was performed by all the participants at SL, HA1, and HA2 on a stool of 16.25 inches. After completion of the test, carotid pulse was measured in a standing position for 15 seconds. The following equation has been used for the calculation of the maximum oxygen uptake capacity:

VO₂ max (ml/kg/min) = 55.23 – (0.09 x pulse rate in beats/min)

Demographic data has been represented as mean ± SD. Physiological and biochemical data have been represented as mean ± SEM. The data analysis was executed using GraphPad Prism software, version 8 (GraphPad, USA). The D’Agostino-Pearson omnibus normality test was used to check the normal distribution of data. A Repeat Measure two-way ANOVA was used to compare the time points of each group from SL with the Bonferroni post hoc test. An unpaired t-test was used to measure the effect of supplementation between the groups at different time intervals. For the statistical significance, a p-value of < 0.05 is considered.

All participants in the Control and supplemented group were similar in age, weight, and height. Table 1 represents the basic characteristics of all the participants. We observe no significant difference in the SBP, DBP, HR, and SpO₂ between the two groups at SL (Table 2). Body composition parameters and VO₂ max values were also similar in both the groups at SL (Table 4 and Figure 2).

Figure 2

Values are represented as mean ± SEM. Statistical significance in control and BRJ supplemented group in comparison to SL measurements is represented as *p < 0.05, **p < 0.01, ***p < 0.001 for each time interval. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 represents the significance between Control and BRJ supplemented group at that particular time interval

Physiological measurements including SBP, DBP, HR, and SpO₂ are summarized in Table 2. Induction to HA affects all the resting parameters. SBP and DBP increased significantly (p < 0.001) in response to HA exposure (HA0) in both the groups as compared to their SL values, thereafter it started decreasing but non-significantly in the Control group. The decrease in SBP and DBP was significant (p < 0.05) in the BRJ supplemented group on HA1 and HA2 as compared to the Control group. A similar response has been observed for resting heart rate in both groups. SpO₂ decrease (p < 0.001) was recorded upon induction to HA in both the groups as compared to SL, which starts improving thereafter. The BRJ supplemented group showed significant amelioration in SpO₂ levels on HA2 in comparison to the Control group.

A slight increase in mean Hb, red blood cells (RBC), and total hematocrit (Hct) was seen in both groups on HA exposure (Table 3). The increase was significant (p < 0.001) in both groups at each time point in comparison to SL. However, the increase in Hb was slightly greater (p < 0.05) in the BRJ supplemented group on HA1 and HA2 as compared to the Control counterpart. No difference was observed for RBC count and total Hct between both groups at any time point.

The body composition was measured by a Bio-electrical impedance analyzer. The results are represented in Table 4. There was a significant decrease in weight and basal metabolic index (BMI) in both groups upon induction to HA. A significant decrease was observed in total body water (TBW) on induction to HA in the Control group while the BRJ supplemented group showed no significant decrease in TBW. The percentage of extra-cellular water (ECW) was found slightly increased after induction to HA in both the groups. Intra-cellular water (ICW) was significantly decreased in the Control group on HA0 and HA1 while the decrease was significant on HA1 in the BRJ group. Although, we observed changes in body composition parameters in both groups as compared to their SL values, but no significant changes were found between both the groups at any time point, which suggests that the changes were due to ascent to HA.

Table 5

(a) Number (percentage) of participants reporting AMS Score on different days at high altitude in the Control group. HAD refers to High Altitude Day and the numbers denotes the day on which AMS score has been recorded
Parameters		HAD1 n (%)	HAD2 n (%)	HAD3 n (%)	HAD4 n (%)	HAD5 n (%)	HAD6 n (%)	HAD7 n (%)
Headache	None Mild Moderate Severe	29 (73) 9 (23) 2 (5) 0 (0)	32 (82) 6 (15) 2 (5) 0 (0)	35 (88) 4 (10) 1 (3) 0 (0)	34 (85) 5 (13) 0 (0) 1 (3)	36 (90) 1 (3) 3 (8) 0 (0)	39 (98) 1 (3) 0 (0) 0 (0)	37 (93) 1(3) 2 (5) 0 (0)
Gastro-intestinal symptoms	None Mild Moderate Severe	38 (95) 2 (5) 0 (0) 0 (0)	39 (98) 1 (3) 0 (0) 0 (0)	40 (100) 0 (0) 0 (0) 0 (0)	38 (95) 2 (5) 0 (0) 0 (0)	37 (92) 3 (8) 0 (0) 0 (0)	37 (92) 3 (8) 0 (0) 0 (0)	38 (94) 1 (3) 1 (3) 0 (0)
Fatigue/weakness	None Mild Moderate Severe	26 (65) 12 (30) 2 (5) 0 (0)	30 (75) 7 (18) 1 (3) 2 (5)	33 (83) 6 (15) 1 (3) 0 (0)	34 (85) 6 (15) 0 (0) 0 (0)	33 (83) 7 (18) 0 (0) 0 (0)	35 (88) 5 (13) 0 (0) 0 (0)	39 (98) 1 (3) 0 (0) 0 (0)
Dizziness/light-headedness	None Mild Moderate Severe	38 (95) 2 (5) 0 (0) 0 (0)	39 (98) 1 (3) 0 (0) 0 (0)	36 (90) 4 (10) 0 (0) 0 (0)	38 (95) 2 (5) 0 (0) 0 (0)	40 (100) 0 (0) 0 (0) 0 (0)	39 (98) 1 (3) 0 (0) 0 (0)	39 (98) 1 (3) 0 (0) 0 (0)
Total AMS Score	None Mild Moderate Severe	35 (88) 5 (13) 0 (0) 0 (0)	36 (90) 4 (10) 0 (0) 0 (0)	38 (95) 2 (5) 0 (0) 0 (0)	39 (98) 1 (3) 0 (0) 0 (0)	40 (100) 0 (0) 0 (0) 0 (0)	39 (98) 1 (2) 0 (0) 0 (0)	40 (100) 0 (0) 0 (0) 0 (0)
(b) Number (percentage) of participants reporting AMS Score on different days at high altitude in the BRJ supplemented group. HAD refers to High Altitude Day and the numbers denotes the day on which AMS score has been recorded
Parameters		HAD1 n (%)	HAD2 n (%)	HAD3 n (%)	HAD4 n (%)	HAD5 n (%)	HAD6 n (%)	HAD7 n (%)
Headache	None Mild Moderate Severe	40 (77) 10 (19) 2(4) 0 (0)	42 (81) 7 (13) 3 (6) 0(0)	48 (92) 4 (8) 0 (0) 0 (0)	46 (88) 5 (10) 1 (2) 0 (0)	47 (90) 4 (8) 1 (2) 0 (0)	49 (94) 2 (4) 1 (2) 0 (0)	48 (92) 4 (8) 0 (0) 0 (0)
Gastro-intestinal symptoms	None Mild Moderate Severe	51 (98) 1 (2) 0 (0) 0 (0)	51 (98) 1 (2) 0 (0) 0 (0)	47 (90) 5 (10) 0 (0) 0 (0)	48 (92) 4 (8) 0 (0) 0 (0)	49 (94) 3 (6) 0 (0) 0 (0)	50 (96) 2 (4) 0 (0) 0 (0)	49 (94) 2 (4) 1 (2) (0)
Fatigue/weakness	None Mild Moderate Severe	41 (79) 9 (17) 1 (2) 1 (2)	40 (77) 11 (21) 1 (2) 0 (0)	43 (83) 9 (17) 0 (0) 0 (0)	41 (79) 10 (19) 1 (2) 0 (0)	44 (85) 6 (12) 2 (4) 0 (0)	41 (79) 10 (19) 1 (2) 0 (0)	45 (87) 5 (10) 1 (2) 1 (2)
Dizziness/light-headedness	None Mild Moderate Severe	50 (96) 2 (4) 0 (0) 0 (0)	51 (98) 1 (2) 0 (0) 0 (0)	52 (100) 0 (0) 0 (0) 0 (0)	50 (96) 2 (4) 0 (0) 0 (0)	51 (98) 1 (2) 0 (0) 0 (0)	48 (92) 4 (8) 0 (0) 0 (0)	50 (96) 2 (4) 0 (0) 0 (0)
Total AMS Score	None Mild Moderate Severe	50 (96) 2 (4) 0 (0) 0 (0)	52 (100) 0 (0) 0 (0) 0 (0)	52 (100) 0 (0) 0 (0) 0 (0)	49 (94) 3 (6) 0 (0) 0 (0)	50 (96) 2 (4) 0 (0) 0 (0)	50 (96) 2 (4) 0 (0) 0 (0)	52 (100) 0 (0) 0 (0) 0 (0)

The results of Queen’s College Step Test for the VO₂ max were represented in Figure 2. HA induction results in a great decrease (p < 0.001) in VO₂ max values in both the group on HA1 and HA2. However, the decrease in VO₂ max values exhibited in the BRJ group is less as compared to the Control group at each time point at HA.

Table 5 (a) and (b) represent the total AMS LLS and score for each symptom of AMS for both groups. Altitude effects on AMS LLS were apparent in both the groups on initial three days but it normalized afterwards. There was no significant difference observed between both the groups. We observed no case of AMS in either group during the study.

We sincerely express our gratitude to the Defense Institute of Physiology and Allied Sciences (DIPAS), Defense Research and Development Organisation (DRDO) for providing funds under the project OPEX (DIP-274). We are also grateful to Director, DIPAS for his constant support and facility.

Karishma Dohare: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Sample collection, Data curation, Writing-original draft. Divya Singh: Data curation, Study planning, Sample collection. Praveen Vats: Conceptualization, Methodology, Data curation, Writing- review and editing, Supervision, and Project administration. Fakir Chand: Sample collection. Subhasis Bose: Sample collection. Rohit Kumar: Sample collection.