This is a single-center, randomized, double-blind, placebo-control clinical trial conducted at Chongqing Xinqiao Hospital and Shigatse Branch Hospital [The Shigatse CARdiorespiratory Fitness (SCARF) Study] in 2022 and registered at www.chictr.org.cn (ChiCTR2200059900 and ChiCTR2200066328). The primary hypothesis of this study is that ubiquinol supplementation may improve cardiorespiratory fitness both at high-altitude acclimatization and de-acclimatization, while AMS and HADAS are secondary endpoints. The study complies with the SPIRIT 2013 recommendations (Standard Protocol Items: Recommendations for International Trials) (24) (Supplementary Material S1). Table 1 depicts the overall schedule and time commitment for trial participants.

Enrolment occurred in Chongqing (low altitude area, 300 m) from June 1st, 2022, and ended on June 15th, 2022. Demographic and medical data of all participants were recorded through interviews. A potential participant must meet all the inclusion criteria and do not meet any exclusion criteria. The inclusion and exclusion criteria are detailed in Table 2. According to previous studies, the risks associated with CoQ10 are minor, even at doses as high as 1,800 mg (25). Moreover, pharmacokinetic studies suggest that exogenous CoQ10 does not influence the biosynthesis of endogenous CoQ10 and is less likely to accumulate in plasma or tissues after the cessation of supplementation (26).

The study protocol complying with the Declaration of Helsinki was approved by the Medical Ethics Committee of Xinqiao Hospital of Army Medical University (No. 2022-060-01). All participants had to provide written informed consent after the study details, procedures, benefits, and risks were explained to them verbally and literally. Failure to do so resulted in their exclusion from this study.

The G-power tool (version 3.1.9.2, Germany, 2017) was used to calculate the sample size by prior power analysis and to estimate all statistically significant results. The α error probability was set at 0.05, and the power (1 − β) was set at 0.9. With a correlation among repeated measures of 0.5, a sample size of 39 was determined to be necessary to assess the effects of ubiquinol. A sample size of 36 within two groups (ubiquinol group and placebo group) and within-between interactions were calculated with a nonsphericity correction (ε) of 1. Considering a 5% dropout rate, based on a sample size of 39, 41 participants would be required finally.

The primary outcome will be the cardiorespiratory fitness characteristics both at high-altitude acclimatization and de-acclimatization after ubiquinol supplementation. Specifically, the parameters including peak oxygen consumption (VO₂), % predicted peak VO₂, VO₂ at the anaerobic threshold (AT), peak O₂ pulse, metabolic equivalents, % maximum predicted heart rate, rate pressure product, VE/VCO₂ slope, oxygen uptake efficiency slope, VO₂/work rate slope, cardiac output, exercise ventilatory power, circulatory power, and dead space ventilation / tidal volume at these four visits will be collected for further analysis. A brief definition of these indicators is as follows: peak VO₂, peak oxygen consumption during an incremental exercise protocol with or without a plateau; % predicted peak VO₂, peak VO₂ divided by predicted peak VO₂; VO₂ at AT, the value of VO₂ at AT time; peak O₂ pulse, the peak oxygen extracted with each beat of the heart; metabolic equivalents, the ratio of exercise metabolic rate to resting metabolic rate calculated as VO₂/3.5 ml/min/kg; % maximum predicted heart rate, heart rate divided by maximum predicted heart rate; rate pressure product, the product of heart rate and systolic blood pressure; VE/VCO₂ slope, from exercise onset to peak exercise by the slope of linear regression of VE and VCO₂; oxygen uptake efficiency slope, slope of the regression line of VO₂ vs. log₁₀ VE; VO₂/work rate slope, ΔVO₂/Δwork rate during incremental exercise; cardiac output, the amount of blood the heart pumps per minute; exercise ventilatory power, the ratio between peak SBP and the VE/VCO₂ slope; circulatory power, the product of CO and mean arterial BP; dead space ventilation / tidal volume, an estimate of the ventilation-perfusion ratio. In addition, the detailed definitions and calculation methods of these indicators are shown in Table 5.

The secondary outcomes will be the questionnaire results of AMS and HADAS. The symptoms of these two diseases are shown in a later section. The effects of altitude and ubiquinol on them will be analyzed in detail in this study. Participants with severe disease symptoms, especially hypoxia-related symptoms, will receive additional treatment.

Figure 1 shows the flow chart of this study. Participants will take ubiquinol orally with a dose of 200 mg daily or a placebo for 14 days before departure. Physical examination, symptoms related to high-altitude acclimatization and de-acclimatization, cardiorespiratory exercise test, laboratory test, and any possible adverse reaction of ubiquinol and exercise will be collected at baseline detection and each visit. During the 14 days of the medication, only participants who did not experience these adverse effects would go to the high altitude as scheduled. It would help us identify related symptoms at high altitudes due to hypoxia rather than adverse events of ubiquinol. The laboratory and exercise tests will be conducted separately on the last three days of the 14 days. Subsequently, they will board a flight to Shigatse (high altitude area, 3,900 m) in 3 h. After arriving at Shigatse, the AMS symptom self-administered questionnaires and adverse reactions of ubiquinol will be followed up daily, and the other prior programs will be carried out on the third day. The subjects will then be flown back to a low altitude after staying for 7 days at a high altitude and scored for HADAS, followed by other prior programs at a low altitude on the 7th day after return. The last visit will be set in the sixth month of this study, which has not yet been conducted. Laboratory tests include a blood routine test of 19 parameters commonly used in the clinic and a serological test of 19 circulating indicators, including glucose, total cholesterol, triglyceride, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, lactate, non-esterified fatty acid, norepinephrine, epinephrine, plasma renin activity, angiotensin Ⅱ, angiotensin-converting enzyme 2, neuropeptide Y, atrial natriuretic peptide, lactate dehydrogenase, insulin, glucocorticoid, adiponectin, and leptin.

Participants will be requested to avoid eating or drinking anything apart from water for up to 12 h. Participants' blood samples will be collected between 8:00 and 8:15 am the day before the exercise tests at different altitudes using the same procedure. After sitting quietly for 15 min, approximately 5 ml of intravenous blood of participants will be collected from the vein at the cubital fossa in the elbow with a tourniquet and mixed immediately with 1 ml of the anticoagulant, dipotassium ethylenediaminetetraacetic acid. Blood samples will be divided into 1 ml and 4 ml parts. The first 1 ml will be analyzed using a BC-3000 plus automated hematology corpuscle analyzer (Shenzhen, China), and the 4 ml will be subjected to high sensitivity enzyme-linked immunosorbent assay kit (Jiangsu Jingmei Biological Technology Co., Ltd., China) for the 19 kinds of circulating parameters. Blood collected at high altitude will be frozen in a −20°C transfer tank and flown to Xinqiao Hospital within 3 h. All blood samples will be measured for biochemical parameters at the Clinical Laboratory of Cardiology Science of Xinqiao Hospital.

The Lake Louise consensus scoring system 2018 version will be used to assess the presence of AMS at high altitudes according to the four main symptoms: headache, gastrointestinal symptoms, fatigue/weakness, and dizziness/vertigo. Scores of 0, 1, 2, and 3 correspond to none, mild, moderate, and severe degrees for each symptom, respectively. AMS is diagnosed as a total score of ≥3, combined with headache (1). We will collect this information every day through self-administered questionnaires at high altitudes.

The HADAS will be evaluated according to the scoring criteria for syndromes, which include complaining of dizziness, fatigue, weakness, headache, blurred vision, lethargy, insomnia, dreaminess, difficulty concentrating, memory loss, slow reaction, and physical signs, including chest tightness, cyanosis, palpitation, slow pulse, precordial pain, changes in blood pressure, decreased appetite, changes in body mass, abdominal distension, diarrhea, edema, constipation, cough, asthma, hair loss, sexual dysfunction, tooth loss, numbness of hands and feet, and skin hemorrhagic spots or ecchymosis (2). In addition, these symptoms do not improve under short-term rehabilitation after organic diseases of the heart, lungs, kidneys, and other organs are ruled out. Physicians who have been systematically trained will score the presence of three or more of the above symptoms or signs according to the following criteria: (1) have a slight reaction but not affect daily work for 0 points; (2) affect work but the symptoms improve soon after rest for score 1; (3) seriously affect daily work and symptoms improve significantly after drug treatment for score 2; (4) seriously affect daily work and symptoms do not improve significantly after drug treatment for score 3.

Participants will be notified in advance to avoid smoking and consuming caffeinated or heavy meals for at least 2 h before the tests. It will be ensured that the indoor environment is suitable for the entire measurement process [in a dedicated temperature-controlled (20–25°C) laboratory]. Lung function will be assessed using the spiroergometry system with Breath-by-Breath technology (Metalyzer 3B, Cortex, Germany, 2022). Standardized assessments of resting lung function will be performed before respiratory tests during exercises. The best attained values of three attempts of forced expiratory volume in one second (FEV1) and forced vital capacity will be recorded. Before each test, the volume sensor will be calibrated using a standard three-liter syringe, and the gas analyzer will be adjusted with two-point calibration using air and a 16.5% O₂:CO₂ mixture. Participants will be provided with a fitting face mask, and volumetric and gas-exchange data will be directly transferred to the metabolic cart software (MetaSoft® Studio, Cortex Biophysik GmbH, 2022), with real-time viewing capacity. Maximum voluntary ventilation (MVV) will be measured from a 12-s maneuver of deep and rapid breathing as the corresponding minute volume. Standards for the performance and interpretation of spirometry data will be obtained from the European Respiratory Society and the American Thoracic Society (42).

Before CPET, standardized assessments of resting lung function will be performed. Whole tests will be conducted in a dedicated temperature-controlled (20–25°C) laboratory by a physician and a skilled technician, who is blinded to other procedures and details of this study. The baseline physiological measures for all devices used in this study will be measured for 5 min in a resting state and subsequently in a standing position. CPET will be performed in an erect position through an electronically braked cycle ergometer (EC3000e, Customed, Germany) with Breath-by-Breath technology (Metalyzer 3B, Cortex, Germany, 2022). The cycle ergometry exercise protocol comprises four stages of exercise: 3 min of rest, 3 min of unloaded exercise, a continual increase in resistance of 25 W/min, and recovery with 3 min of unloaded cycling. All patients will continue CPET until the development of limiting symptoms such as angina, dyspnea, or muscular fatigue. CPET will be terminated at the discretion of the operating physician if pre-specified termination criteria develop, such as hemodynamic abnormalities, heart rhythm abnormalities, or neurological impairment.

The AT time is estimated from breath-by-breath data using the V-slope method and validated with other plots using the ventilatory-equivalent method and the respiratory exchange ratio method (31, 43). The parameters for AT will be obtained at this time point. Other peak values will be calculated at identical time points. The calculation method for each indicator detected using the metabolic cart software (MetaSoft® Studio, Cortex Biophysik GmbH, 2022) is described in detail in Table 5.

Standard 12-lead electrocardiogram, blood pressure, and SpO₂ will be obtained throughout the procedure using a 12-lead connection (Custo-Cardio 3000BT-A, Cortex, Germany, 2022) in real-time, blood pressure cuffs (Suntech Tango M2, Cortex, Germany) in the upper arm and a portable finger clip oximeter (Nonin wristOx2 3150, Nonin, United States), respectively. All these parameters of CPET are the direct output from the cardiopulmonary exercise testing system.