Low Oxygen Exposure and Weight Status

Body weight will be measured during the baseline weight maintenance phase (days -14 to 0) and each morning during the study (days 1 to 56) following an overnight fast and morning void, using a calibrated digital scale provided to participants to use at home (A&D Medical wireless weight scale UC-352BLE, San Jose, CA). Change in body weight will be calculated as: body weight on day 56 - baseline body weight (average of days -14 to 0).

Body composition will be determined following at least a 10 hour fast and morning void on day -7 and 49 using dual energy x-ray absorptiometry (DEXA, Discovery W, Hologic Inc., Bedford, MA or equivalent). The volunteer will remove all metal-containing objects from their body, lay face-up on the DEXA densitometer table in light clothing (t-shirt, shorts, socks), and be asked to remain motionless for the 8-10 min scan. A scanner emitting low energy x-rays and a detector will pass over the participant. These data will be used to calculate total body mass, fat-free mass, fat mass, and bone mass. Total body water will be measured following at least a 10 hour fast and 1 hour without water intake using oxygen-18 labeled water on days -7 and 49.

Waist circumference will be measured to the nearest centimeter using a measuring tape. Measurements will be taken in duplicate.

Blood volume will be determined using the optimized carbon monoxide rebreathing method on days -7 and 49. In this method, a finger stick blood sample is taken and basal carboxyhemoglobin concentrations determined using a blood gas analyzer (OSM-3, Radiometer). Carbon monoxide is then inhaled via a spirometer (SpiCo, Blood Tec.) for 2 min. Seven minutes after inhaling the carbon monoxide bolus, a second finger prick blood sample is taken to determine the increase in carboxyhemoglobin concentration. The change in percentage of carboxyhemoglobin between the basal and second measurements can be used to calculate hemoglobin mass and blood volume.

Energy expenditure will be assessed by the doubly labeled water (DLW) method. Briefly, upon waking, volunteers will provide a urine sample and completely void their bladder, this sample will be used to measure background isotope enrichment. The participant will then consume 1.5 g/kg of 10% O18 and 0.06 g/kg of deuterium. Urine samples will be collected at 4 h, 6.5 h, 24 h, 3 d, 5 d, and 7 d after dosing.

Resting metabolic rate will be measured with open circuit, indirect calorimetry (ParvoMedics TrueOne 2400 metabolic cart, Sandy, UT) in the morning, upon arrival to the lab on days 0 and 56.

Resting substrate oxidation will be measured with open circuit, indirect calorimetry (ParvoMedics TrueOne 2400 metabolic cart, Sandy, UT) in the morning, upon arrival to the lab on days 0 and 56.

Participants will be provided an ad libitum mixed-macronutrient meal (e.g., frozen lasagna) on days 0 and 56. Food will be provided in excess of expected consumption for one individual. Water in the amount of 240 g will be provided during the meal. Participants will be instructed to consume all water before completing the meal and will not be permitted additional water during the meal, in order to control the effect of fluid intake on satiety. To limit social influence on eating behavior, each ad libitum meal will be consumed in isolation. Leftover food will be collected and weighed after participants have left the laboratory. Energy and nutrient content of the amount consumed will be determined using Food Processor software (version 11.6.0, ESHA Research, Salem, OR) under the supervision of a Registered Dietitian.

Subjective appetite will be measured on study days 0 and 56. Subjectively rated appetite will be assessed immediately before the oral glucose tolerance test (OGTT), at 15, 30, 45, 60, 90, and 120 minutes after beginning the OGTT, and upon cessation of the ad libitum meal (discussed above) using 100 mm visual analog scales to rate fullness, hunger, desire to eat, and prospective food consumption. Composite satiety score will be calculated from the individual appetite scores using the equation: CSS = (fullness + (100 - Desire to eat) + (100 - Hunger) + (100 - Prospective food consumption) / 4.

Participants will be provided a wrist-worn device with Bluetooth capabilities (vivosmart 4, Garmin, Olathe, KS) to be worn for 2 weeks prior to the start of the study (baseline measurement) and continuously for the entirety of the study. This devise will measure steps, physical activity levels, heart rate, overnight oxygen saturation, and sleep patterns.

In addition to the wrist-worn physical activity monitoring device (vivosmart 4, Garmin, Olathe, KS) that will measure overnight oxygen saturation, participants also will wear a pulse oximeter (PalmSAT® 2500A) on their index finger overnight to measure oxygen saturation.

The prevalence and severity of AMS will be determined from information gathered using the shortened version of the Environmental Symptoms Questionnaire (ESQ) and the Lake Louise AMS Scoring System (LLS; 5 min; 26 questions total). At completion of the questionnaire, oxygen saturation will be determined using finger pulse oximetry (PalmSAT® 2500A, Nonin Medical, Inc., Plymouth, MN). AMS and oxygen saturation measurements will be taken in the morning (between 5:00-8:00am, inside tent) and evening (9:00pm-12:00am, inside tent) on days 0-7, 14, 21, 28, 35, 42, and 56.

A 3-lead electrocardiogram will be conducted on days 0 and 56 to assess heart rate variability. Participants will have electrode patches placed on their chest to measure the electrical activity of the heart.

Participants will be provided a wrist-worn device with Bluetooth capabilities (vivosmart 4, Garmin, Olathe, KS) to be worn for 2 weeks prior to the start of the study (baseline measurement) and continuously for the entirety of the study. This devise will measure steps, physical activity levels, heart rate, overnight oxygen saturation, and sleep patterns.

On days -1, 13, and 55 urine will be collected during the overnight time period. Participants will void their bladder before entering the tent in the evening (they will not collect this portion). Participants will collect any urine produced throughout the night and will collect one final time in the morning after exiting the tent. Epinephrine concentrations in the urine samples will be measured.

On days -1, 13, and 55 urine will be collected during the overnight time period. Participants will void their bladder before entering the tent in the evening (they will not collect this portion). Participants will collect any urine produced throughout the night and will collect one final time in the morning after exiting the tent. Norepinephrine concentrations in the urine samples will be measured.

On days -1, 13, and 55 urine will be collected during the overnight time period. Participants will void their bladder before entering the tent in the evening (they will not collect this portion). Participants will collect any urine produced throughout the night and will collect one final time in the morning after exiting the tent. Dopamine concentrations in the urine samples will be measured.

A 75-g oral glucose tolerance test (OGTT) will be conducted on days 0 and 56. A venous catheter will be placed and blood samples obtained at -5, 30, 60, 90, and 120 min during the OGTT for measurements of plasma glucose and insulin concentration. Insulin sensitivity will be determined by using the whole-body insulin sensitivity index (WBISI), also known as the Matsuda Index.

A 75-g oral glucose tolerance test (OGTT) will be conducted on days 0 and 56. A venous catheter will be placed and blood samples obtained at -5, 30, 60, 90, and 120 min during the OGTT for measurements of plasma glucose and insulin concentration. Insulin sensitivity will be determined by using the whole-body insulin sensitivity index (WBISI), also known as the Matsuda Index.

A 75-g oral glucose tolerance test (OGTT) will be conducted on days 0 and 56. A venous catheter will be placed and blood samples obtained at -5, 30, 60, 90, and 120 min during the OGTT for measurements of plasma glucose and insulin concentration. Insulin sensitivity will be determined by using the whole-body insulin sensitivity index (WBISI), also known as the Matsuda Index.

The thermic effect of glucose will be measured on study days 0 and 56 with open circuit, indirect calorimetry (2400 TrueOne, Parvo Medics) using established protocols. Resting metabolic rate measurements will be taken prior to glucose administration in a thermo-neutral room while the participant is in the supine position (as described above). During the oral glucose tolerance test the thermic effect of glucose will be measured in 15 min increments for 2 hours (15-30, 45-60, 75-90, and 105-120 minutes).

A continuous glucose monitor (Freestyle Libre Pro) will be inserted into the subcutaneous adipose tissue behind the arm and will be used to collect and assess 24-hour glucose concentrations.

A continuous glucose monitor (Freestyle Libre Pro) will be inserted into the subcutaneous adipose tissue behind the arm and will be used to collect and assess 24-hour glucose concentrations.

Participants will be provided a wrist-worn device with Bluetooth capabilities (vivosmart 4, Garmin, Olathe, KS) to be worn for 2 weeks prior to the start of the study (baseline measurement) and continuously for the entirety of the study. This devise will measure steps, physical activity levels, heart rate, overnight oxygen saturation, and sleep patterns.

The Pittsburgh Sleep Quality Index will be used to examine participant sleep quality on days -1, 14, and 56 (5 min; 18 questions total).

Participants will be asked to complete a sleep diary before and after sleep for 7 d at baseline, on days 0-14, and on days 49-56. The sleep diary includes questions related to mood, caffeine intake, substance use (e.g. alcohol, caffeine, and prescriptions), and sleep quality.

Serum iron, total iron binding capacity, soluble transferrin receptor, hepcidin, hemoglobin, hematocrit, ferritin, erythroferrone, and erythropoietin will be measured in fasting blood samples collected on days 0 and 56.

The Positive and Negative Affect Schedule (PANAS), Cohen Perceived Stress Scale, Big Five Inventory-2, Delay Discounting questionnaire, Palatable Eating Motives Scale, and the Generalized Self-Efficacy scale will be administered on day 0, 14, and 56.

Participants will rest in a seated position for at least 5 minutes before an automated blood pressure cuff is used to measure blood pressure in triplicate.

This questionnaire will be administered to participants on day 56 to assess real and perceived barriers to sleeping in a hypoxic tent (5 min; 17 questions total).

Body weight also will be measured 4 weeks after the study intervention ends to assess whether weight loss is sustained. Change in body weight will be calculated as:

• Body weight on day 84 (i.e., 4 wk post-intervention) - baseline body weight (average of days -14 to 0)
• Body weight on day 84 (i.e., 4 wk post-intervention) - body weight on day 56