Comparison of Bi-environmental Conditions During Occupational Related Activity

Each participant will perform 5 study visits: 1) baseline testing and four trials.

Participant numbers will be randomly assigned. Four trials for men (extreme cold, EC; Moderate Cold, MoC; mild cold, MiC; extreme heat, EH) and four trials for women (EC-follicular phase, EC-F; EC- luteal phase, EC-L; EH-F; EH-F) will be ordered with a random number generator and assigned to a participant number. These orders will be counterbalanced between participants to ensure that there is not an order effect. Participants will be assigned after medical clearance is granted. Visit specific information is described below, however, specific data collection protocols for dependent variables and procedures are included below the description of the study design. Participants will be asked to sign a photo/video release form (Appendix H) to be used in research projects, scientific publications/conferences and for educational purposes. Participants may be videoed or photographed while exercising or resting in the environmental chamber.

Baseline Testing (visit 1)

Participants who are approved to participate will be scheduled for a baseline and VO2max

testing day. Upon arrival to the lab, subjects will provide a urine sample in a clean urine cup.

Urine specific gravity and urine color will be assessed. Nude body mass will also be recorded. Privacy will be afforded for each participant while obtaining body mass measures by taking measurements with the participant alone in a private room behind a solid, closed door, and the research reading the scale outside of the room. Participants will only be permitted to continue to

the VO2max test if the euhydrated criteria of a urine specific gravity (USG) ≤1.025 is met. If the participant arrives with a USG between 1.020 and 1.025, the subject will be asked to drink 500mL of water to ensure proper hydration status. Participants will fill out a Karolinska Sleep Diary (Appendix I) prior to collecting anthropometrics. Females will fill out a menstrual history questionnaire (Appendix K). Height will be measured with a tape measure. Percent body fat will be measured using Air displacement plethysmography (BodPod®; Cosmed, Software Version 4.2+, Concord, CA, USA). This will take less than ~5 minutes to complete. This information will be used to describe the subject demographics. For familiarization purposes, researchers will insert and immediately remove the esophageal probe. Participants will also be asked to complete a maximal oxygen uptake (VO2max) test on a treadmill in a thermoneutral environment. This test will provide aerobic fitness levels of the participants. After VO2max testing, researchers will assess the 6 locations where blood flow analyses will occur during trial days (described in the dependent variable section). This will familiarize the participant to the blood flow process and allow researchers to optimally location each artery of interest prior to the testing days. Researchers may also use a sharpie or tracing paper to mark anatomical landmarks to assist with artery identification during trial days. This visit will take approximately 1-2 hours.

Substudy #1 Males and Females in Extreme Cold and Heat

This substudy will have two visits for males and four visits for females (figure below).

Participants will perform 30 mins of rest outside the climate chamber, one hour of rest inside the climate chamber, and 2 hours of work to rest cycles in the climate chamber.

Participants will be allowed to make-up trial visits if they are sick, suffer an injury or do not meet hydration requirements. Participants will be asked if they have a current illness or taking medications to determine whether they can complete the trial. The participant's visit will be rescheduled if they have a fever, current illness or taking medications that influence body temperature. Both trials will be on separate days at least 3 days apart. Women will determine their phase of their menstrual cycle based on self-reporting and a menstrual history questionnaire filled out during the baseline visit (Appendix K). The EC and EH trials in the follicular phase will be collected 14 days following the start of menses and the luteal phase will occur between 14 and ~30 days of cycle.

Upon arrival to the lab, subjects will provide a urine sample in a clean urine cup. Urine specific gravity and urine color will be assessed. Nude body mass will be recorded. Privacy will be afforded for each participant while obtaining body mass measures by taking measurements with the participant alone in a private room behind a solid, closed door, and the research reading the scale outside of the room. Participants will only be permitted to continue to the trial if the euhydrated criteria of a urine specific gravity (USG) ≤1.025 is met. If the participant arrives with a USG between 1.020 and 1.025, the subject will be asked to drink 500 mL of water to ensure proper hydration status.Participants will be given instructions at the prior visit about how to obtain desired hydration status for the upcoming trial.

Upon arrival to the lab, participants will also be asked to indicate if they have consumed alcohol or caffeine in the last 24 hours and 12 hours, respectively. They will not be asked to specify exactly what they have consumed, but if they provide a positive indication, their trial will be rescheduled for another day. Key study personnel will insert an esophageal probe to measure esophageal temperature (procedure described below). No undergraduate student who is listed as key personnel will insert an esophageal probe Participants will privately insert a rectal thermometer 15 cm beyond the anal sphincter and wear a heart rate monitor strap. Additionally, participants will wear 14-site skin temperature sensors (Biopac). Researchers will apply the skin temperature sensors with medical tape to the participants' finger tip and base of finger, hand dorsum, forearm, thigh, calf, feet dorsum, big toe, nose, cheek, ear tip, chin, and forehead. Participants will also have 4 iButton skin temperature sensors placed on four locations (shoulder, chest, calf, thigh) to assess mean skin temperature. The participants will also be asked to complete a subjective sleep questionnaire upon arriving to the lab. the participants will wear a Samsung Gear S3 smartwatch on their non-dominant arm and a Polar chest strap. These devices would be paired with a Samsung Galaxy Note 4 smartphone that runs the 2B-Cool algorithm for core temperature estimation using data from the smartwatch. These devices would be provided by the BHSAI to the University of Connecticut.n addition to the BHSAI-provided devices, participants will wear an apple watch and garmin watch on the opposite hand. Both of these devices will be validated against the gold standard assessments (Table 2). Each dependent variable coming from the Apple and Garmin devices from the manufacturer's own applications. All measurements will be taken starting from the thermoneutral exposure (pre-exposure) period to include baseline recordings. Blood flow of the brachial artery, radial and ulnar artery, superficial femoral artery, anterior tibial artery, and posterior tibial artery will be collected with a Triplex ultrasound during rest (before exercise and during rest breaks). The same sequence for the measuring sites across subjects, e.g., 1) radial, 2) ulnar, 3) brachial, 4) femoral, 5) anterior tibial, and 6) posterior tibial, will be used. A pulse oximeter will also be clipped to the participant's finger and measured every 10 mins throughout each rest period. Blood lactate will be assessed from a finger stick blood sample (0.7μ1) during exercise trials and post-exercise. VO2 data will also be assessed to collect metabolic heat production. After thermoneutral exposure of 30 minutes, participants will rest in a lounge chair for an hour and then perform 2 hours of moderate exercise with rest breaks. All dependent variables listed above will be collected through the exercise period at various time points. Participants will drink ad libitum throughout the exercise trial. Participants will be asked to keep a dietary record (using My Fitness Pal) to track their food and fluid intake during the trial period.

Extreme Cold Trial

During the EC trial, participants will wear a t-shirt, shorts, a microfleece cap and lightweight balaclava. Participants will also wear ankle length socks (no shoes during rest). Participants will rest upright on a chair with feet above the ground and hands on the arms of the chair (exposed to air). The participant will remain seated for up to 1 hour. For additional safety of the participants, the experiment will be terminated if the core body temperature falls below 35°C. Participants will wear t-shirt, shorts, microfleece cap, and shoes for the exercise component of the protocol. The trial will be conducted outside the chamber (~20C) and in a climatic chamber with ambient temperature below <8°C and relative humidity at approximately 30%.

Extreme Heat Trial

During the EH trial, participants will wear a t-shirt and shorts, a microfleece cap and lightweight balaclava during rest. Participants will also wear ankle length socks (no shoes during rest). Participants will remove the cap and balaclava prior to exercise. During rest, participants will rest upright on a chair with feet above the ground and hands on the arms of the chair (exposed to air). The participant will remain seated for up to 1 hour. For additional safety of the participants, the experiment will be terminated if the core body temperature exceeds 40C. Participants will wear t-shirt, shorts, microfleece cap, and shoes for the exercise component of the protocol. The trial will be conducted outside the chamber (~20C) and in a climatic chamber with ambient temperature between 30-42C and relative humidity at approximately 30-60%. Participants will also complete an environmental symptoms questionnaire (ESQ).

Substudy #2 Males Moderate and Mild Cold Stress at Rest

Participants will perform 30 mins of rest outside the climate chamber and two hours of rest inside the climate chamber in moderate vs. mild cold (Figure 3). The data collection procedures are summarized below and in Figure 3. The subjects will be male only and will the same male subjects participating in substudy #1.

Trial Day Procedures (moderate and mild cold).

Participants will be allowed to make-up trial visits if they are sick, suffer an injury or do not meet hydration requirements. Participants will be asked if they have a current illness or taking medications to determine whether they can complete the trial. The participant's visit will be rescheduled if they have a fever, current illness or taking medications that influence body temperature. Both trials will be on separate days at least 3 days apart. Upon arrival to the lab, subjects will provide a urine sample in a clean urine cup. Urine specific gravity and urine color will be assessed. Nude body mass will be recorded. Privacy will be afforded for each participant while obtaining body mass measures by taking measurements with the participant alone in a private room behind a solid, closed door, and the research reading the scale outside of the room. Participants will only be permitted to continue to the trial if the euhydrated criteria of a urine specific gravity (USG) ≤1.025 is met. If the participant arrives with a USG between 1.020 and 1.025, the subject will be asked to drink 500 mL of water to ensure proper hydration status.

Upon arrival to the lab, participants will also be asked to indicate if they have consumed alcohol or caffeine in the last 24 hours and 12 hours, respectively. They will not be asked to specify exactly what they have consumed, but if they provide a positive indication, their trial will be rescheduled for another day. Key study personnel will insert an esophageal probe to measure esophageal temperature (procedure described below). No undergraduate student who is listed as key personnel will insert an esophageal probe Participants will privately insert a rectal thermometer 15 cm beyond the anal sphincter and wear a heart rate monitor strap. Additionally, participants will wear 14-site skin temperature sensors. Researchers will apply the skin temperature sensors with medical tape to the participants' finger tip and base of finger, hand dorsum, forearm, thigh, calf, feet dorsum, big toe, nose, cheek, ear tip, chin, and forehead. Participants will wear a 3-lead ECG device. The participants will also be asked to complete a subjective sleep questionnaire upon arriving to the lab. the participants will wear a Samsung Gear S3 smartwatch on their non-dominant arm and a Polar chest strap. These devices would be paired with a Samsung Galaxy Note 4 smartphone that runs the 2B-Cool algorithm for core temperature estimation using data from the smartwatch. These devices would be provided by the BHSAI to the University of Connecticut. All measurements will be taken starting from the thermoneutral exposure (pre-exposure) period to include baseline recordings. Blood flow of the brachial artery, radial and ulnar artery, superficial femoral artery, anterior tibial artery, and posterior tibial artery will be collected with a Triplex ultrasound during rest (before exercise and during rest breaks). The same sequence for the measuring sites across subjects, e.g., 1) radial, 2) ulnar, 3) brachial, 4) femoral, 5) anterior tibial, and 6) posterior tibial, will be used. VO2 data will also be assessed to collect metabolic heat production. A pulse oximeter will also be clipped to the participant's finger and measured every 10 mins throughout each rest period. Blood lactate will be initially assessed using a finger stick blood sample (0.7 μ1) during the baseline period (thermoneutral). After thermoneutral exposure of 30 minutes, participants will rest in a lounge chair for two hours. All dependent variables listed above will be collected through the exercise period at various time points. Participants will drink ad libitum throughout the exercise trial. Participants will be asked to keep a dietary record (using My Fitness Pal) to track their food and fluid intake during the trial period. Participants will wear a t-shirt, shorts, and microfleece cap. Participants will also wear ankle length socks (no shoes). Participants will rest upright on a chair with feet above the ground and hands on the arms of the chair (exposed to air). The participant will remain seated for up to two hours. For additional safety of the participants, the experiment will be terminated if the core body temperature falls below 36°C. The trial will be conducted outside the chamber (~20C) and in a climatic chamber for moderate cold will have an ambient temperature between 8-10°C and mild will be 10-15C (relative humidity at approximately 30% for both).