Rehydration With a Trace Mineral Supplement

Protocol Before the start of the study eligible participants will visit our lab to conduct a screening of medical history, weight, height, and a onetime assessment of body composition via BIA and Training history. This visit will take place at least one day before the actual start of the study and typically lasts no longer than 30 minutes. During this visit participants will be given two questionnaires, one regarding the physical activity readiness of the participant, and another one regarding their health history. If a participant does not meet the physical criteria and does not pass both questionnaires, they will be given a $25 digital Tango gift card for their brief visit to our lab. Participants who do pass will be given a scheduled date and time to visit the lab again for their first official trial.

At the start of the study subjects will visit the lab after at least 10h of fast and they will consume a light breakfast. An intravenous catheter will be place in an antecubital vein and a blood sample will be taken after 20 min for baseline. A baseline skin hydration reading will also be taken at this time. Subjects will void their bladders and a body weight will be taken.

Dehydration:

Consistent with the study by Shirreffs et al (2007) and based on our previous work, dehydration will be accomplished on the morning of testing via mild exercise and heat exposure to elicit a body weight loss of -2%. The subjects will perform four bouts of 25-minutes low-intensity exercise and 5 min rest (alternating cycling \& walking) or till they reach -2% of body weight dehydration.

Rehydration:

Upon losing 2% body weight, participants will be instructe to rinse off their face to get rid of excess sweat. This is followed by a 20 minutes rest period in a thermo-comfortable environment, a dehydrated baseline blood sample will be taken, along with a dehydrated skin hydration reading and subject will start the rehydration protocol.

The volunteers will consume one of the three drinks below in a cross over balance mode. During the first hour four equal doses will be consumed in15-minutes intervals at a total volume of 150% of their body weight loss.

Trials/Drinks (block randomization will be used to assign individual participants to each specific trial/drink using their ID number):

• Water
• ConcenTrace®
• ZEROLyte™ Note: The beverage volumes will be standardized at 150% of body weight loss during exercise-induced dehydration to ensure consistency across trials.

See the study design expressed as a figure below:

Measurements:

All measurements will be performed at one of the test rooms at our lab facility that boarders the room where participants can relax and wait out the end of the study day. When certain measurements need to be performed they simply move from one room to the next. Duration of taking blood and urine samples will take not more than 30-90 seconds each and perceptual data will take 30-60 seconds to be reported. To allow to perform all measurements, participants will remain supervised by a team member in the lab for up to 7 hours per trial. Therefore, there will be a separate space available that allows for self-study or watching TV that includes a chair and table to work on and a couch to relax.

Blood samples (7 per trial):

Blood samples will be taken via a venous catheter attached to an extension tube and a stopcock. After each blood sample sterile saline solution will be infused in the extention tube to avoid clotting. The iv catheter will be placed by one of the Clinical Research Center phlebotomists. A total of seven 10 milliliters blood samples will be collected on the euhydrated baseline, dehydrated baseline and at 30, 60, 120, 180, and 240 minutes of the rehydration period.

All samples will be analyzed for glucose, osmolality, total plasma protein, hematocrit, hemoglobin (for plasma volume changes), sodium, potassium, and chloride.

Urine samples (6 per trial):

Urine samples will be provided from the subjects in a urine cup. The cup will be placed in the pass-through window where a lab technician will receive it and process it.

Urine samples will be collected using \~900 milliliters cups at euhydrated baseline, dehydrated baseline and cumulative totals will be collected at 60, 120, 180, and 240 minutes of the rehydration period. All samples will be analyzed for urine osmolality, specific gravity, volume, sodium, and potassium.

Sweat samples (1 per trial):

Sweat samples will be collected with sweat patches. The sweat patches are like a band-aid that it will be placed on the forearm and stay there for approximately 30 min to absorb sweat. After 30 minutes the patch will be removed and the sweat sample will be extracted by centrifugation in the lab.

Sweat samples will be collected during the first 30 min of exercise to assess sodium and potassium content.

Skin Hydration (6 per trial):

Skin hydration will be measured with a pen-like instrument that applies a very light pressure on the skin. This does not cause any pain or skin irritation.

Skin hydration will be measured on the face (inner zygomatic bone), arm (bicep brachii) at euhydrated baseline, 30 minutes after the end of dehydration, and at 60, 120, 180, and 240 minutes of rehydration.

Gastrointestinal Symptoms (6 per trial):

Gastrointestinal symptoms will be assessed at 1, 2, 3 and 4 hours of rehydration via Gastrointestinal Symptom Rating Scale (GSRS).

Perceptual Data (6 per trial, maximal 9 minutes):

Thirst, stomach fullness, and taste, assessed via a visual analog scale.