The goal of this clinical trial is to learn if one week of creatine monohydrate supplementation alters inflammation, markers of acute kidney injury (AKI) risk, and cognitive performance in active, young, healthy volunteers. The main questions it aims to answer are:

1. Does one-week of creatine supplementation alter urinary markers of inflammation (cytokines) during exercise in the heat when compared to placebo?
2. Does one-week of creatine supplementation alter markers of AKI during exercise in the heat when compared to placebo?
3. Does one-week of creatine supplementation attenuate reductions in cognitive measures as a response of fatigue from exertional heat stress.

Researchers will compare creatine monohydrate to a placebo (maltodextrin) to see if it alters inflammation, risk of acute kidney injury, and cognition after exercise in the heat.

Participants will :

• Complete a baseline aerobic exercise test to measure VO2max
• Ingest 20 grams per day of creatine monohydrate or placebo (maltodextrin) for seven consecutive days
• Cycle for 90 minutes in a heated chamber (38 C, ~40% relative humidity), alternating low- and high-intensity exercise
• Self-collect urine before, after, and one-hour after exercise
• Self-insert a rectal thermistor to measure core temperature
• Complete cognitive tasks using the NIH toolbox before and after exercise to assess cognition
• Complete the other condition (placebo or creatine) at least 21 days later

The outlined research questions/ hypotheses will require 3 visits (1 baseline, then 2 experimental trials) to the UNI Exercise Physiology Lab.

The first (baseline) visit will involve completing paperwork (informed consent, health history questionnaire, physical activity readiness questionnaire; PAR-Q+), familiarization of cognitive assessment techniques, anthropometric assessments, and a maximal effort, graded exercise test.

Baseline Visit:

- Height and weight will be measured using a standing stadiometer and InBody bioimpedance analyses full body analyzer

• Baseline/ Familiarization Cognitive Test: The NIH Toolbox Fluid Cognition Battery and Color Word Stroop Test (CWST) will be administered for baseline cognitive values and familiarization. This will be administered via iPad in a quiet room with minimal distractions
• Cycle Ergometer Test: Aerobic fitness will be assessed using a graded, maximal oxygen consumption test on a cycle ergometer. Subjects will be given a nose clip and mouthpiece which will be connected to a metabolic cart. Heart rate response and gas exchange will be monitored throughout the test.
• Identification of workloads used for experimental trials: Following the cycle ergometer test, participants will rest in a seated position for 10-minutes. They will then undergo a series of 3-minutes submaximal stages to identify workloads which correspond to an estimated metabolic rate of 4 Watts/ kilogram of body weight (4 W/kg) and 8 W/kg. Watts will be estimated via measuring gas exchange (VO2 and Respiratory exchange ratio). These intensities were chosen as they were the average and peak rates which have been observed in a sample of construction workers during work in the summer.

Exercise Visits (Creatine and Placebo, Visits 2 & 3):

Each experimental exercise visit consists of supplementation with a standardized breakfast and 90-minutes of exercise on a stationary cycle ergometer located in the heat chamber of the Exercise Physiology Lab. To ensure participant safety and consistency in data collection, the following outlines the environmental conditions, safety considerations, exercise protocols, and measurement procedures that will be followed for each exercise visit.

Environmental Conditions:

All exercise testing will occur in a controlled environmental chamber set to simulate a hot exercise environment. Conditions within the chamber will be maintained at 38 °C (100.4 °F) and 40% relative humidity (WBGT: ~30.2 °C) and monitored continuously via Kestrel Drop temperature and humidity monitoring device.

Supplement Administration:

1. During the 7 days prior to each exercise visit, the participant will be asked to ingest 20 g/ day of creatine or placebo in a blinded fashion. This will be taken four times per day in 5 grams of supplement ingested with water.
2. At least one week prior to the trial, a member of the research team will provide the participant with 28 bags of 5 grams of creatine or placebo.

Exercise Visits (visits 2 and 3):

1. Upon arrival, participants will provide a urine sample for hydration assessment (subjects will collect their own urine sample, in private one-room bathroom located in Exercise Physiology Lab).

2. Urine Specific Gravity (USG) will be measured using a refractometer following standard procedures to confirm hydration.

   4. Bioelectrical Impedance Analysis: Participants will step on an InBody device which will measure % body fat.

   5. Sleep questionnaire: Participants will be asked to fill out a sleep questionnaire to get an idea if creatine altered sleep quality or duration.

   6. Standardized breakfast, water, and supplement Administration (1 hour prior to exercise)

a. Once adequate hydration is confirmed, participants will consume the assignment supplement (5 grams of creatine or placebo) with a standardized breakfast. After full consumption of food and supplement, the research team will start the 1-hour timer to ensure that a full 60-minutes is waited before starting exercise.

7. After Supplementation but Prior to Exercise:

1. Cognition: Participants will complete a baseline Color Word Stroop Task and NIH Toolbox Fluid Cognition Battery test via Ipad in quiet and distraction free environment.
2. Blood Pressure and Blood collection:

i. Participants will sit for 10-minutes. ii. Blood pressure will be recorded and measured. iii. A capillary blood sample will be taken from the earlobe or fingertip to measure blood composition.

c. Urine Collection: In a private restroom, participants will void their bladder into a beaker. Urine will then be stored at -80 °C until later analyses for markers of acute kidney injury.

d. Nude Body Weight: Participants will be instructed to enter a private bathroom to measure nude body weight and self- insert a rectal thermistor to measure core temperature: e. Monitoring Devices: i. Participants will be fitted with a Polar chest-strap that contains a heart rate monitor.

ii. Participants will be fitted with 4, non-invasive skin temperature iButton sensors, placed on the chest, arm, thigh, and leg.

8.Pre-Exercise Baseline Measures

1. Participants will enter the heat chamber and sit in a chair
2. While seated, the following baseline measures will be collected:

i. Profile of Mood States (POMS) Questionnaire ii. Color-Word Stroop Test (CWST) (a cognition test) iii. Thermal Comfort & Strain Scale (TCS) (to understand perceived heat stress) iv. Rating of Perceived Exertion (RPE) v. Heart Rate (HR) and Core Temperature (Tc) vi. Room Temperature and Humidity vii. Visual Analogue Scale for Fatigue (VAS-F) viii. Gastrointestinal Symptoms Questionnaire (GIQ) ix. Environmental Symptoms Questionnaire (ESQ)

9. Exercise Protocol

1. Participants will cycle on a stationary bike for 90-minutes at a pre-determined intensities corresponding to workloads of 4W/kg and 8 W/kg, as determined by VO2max test. Specifically, the participant will complete 3, 30-minute cycles consisting of 20-minutes at 4 W/kg and 10-minutes at 8 W/kg. Participants will be asked that they not drink water during the trial, but water will be available if participants want it.
2. HR and Tc will be continuously monitored throughout exercise
3. At every 10 minutes, the following measures will be collected and recorded:

i. HR ii. Tc iii. RPE iv. TCS v. Room Temperature and Humidity vi. Visual Analogue Scale for Fatigue (VAS-F) d. At the end of each 30-minute cycle, participants will complete the CWST while cycling.

e. During minutes 15-25 of each 30-minute cycle (the last 5-min at 4 W/kg and the first 5-min at 8 W/kg), the participant will be hooked up to the same mouthpiece and nose clip used for baseline aerobic testing. This will be used to calculate estimated metabolic rate during the trial.

10. Post-Exercise Heat Measures (in heat chamber):

1. After completing the 90-minute exercise protocol, participants will remain seated in the chamber for 10-minutes
2. Starting at the 5-minute post-exercise mark, the following will be collected:

i. HR ii. Tc iii. RPE iv. TCS v. CWST vi. Room Temperature and Humidity vii. Visual Analogue Scale for Fatigue (VAS-F) viii. POMS ix. GIQ x. ESQ xi. Blood Pressure xii. I-STAT capillary blood measurement

11.Post-Exercise Hydration Assessments

1. Participants will provide a urine sample to reassess hydration using USG, urine flow rate, and store urine for future analyses (as described above).

2. They will also obtain a second nude body weight following the same procedure as pre-exercise.

3. They will also provide a urine sample.

4. Following this, participants will be given a standardized volume of water (500 mL for individuals < 75 kg, 1 L for individuals ≥75kg) to ingest by the end of the hour following the end of the exercise trial.

5. During this time, participants will be allowed to change clothing

   12. After providing urine and changing, participants will complete all cognition assessments.

   13. 1-Hour Post-Exercise Hydration Assessments

a. Another blood collection measurement will be conducted. b. Participants will provide a urine sample to reassess hydration using USG, urine flow rate, and store urine for future analyses.

14. Subjects will then be scheduled for their second exercise visit (following 28-day washout period- 21 day washout then 7 days of supplementation).

Inclusion Criteria:

• Age range: 18-45 years old
• Physical Activity Level: Classified as 'active' using the criteria of engaging in planned exercise at least 3x/week, for a minimum of 30 minutes per session, over the past 3 months
• Aerobic Fitness Level: Subjects must have an aerobic fitness classification of at least 'good' per American College of Sports Medicine (ACSM) criteria and assessed via maximal oxygen uptake test
• Health Status: Free of any known cardiovascular, metabolic, renal, or neurologic conditions.
• Physical Limitations: Indicates no physical limitations to cycling for ~ 90 minutes
• Supplementation: Reports no use of creatine within the past 4 weeks
• Recent Illness: Reports no presence of illness, vomiting, or diarrhea in the previous three days.

Exclusion Criteria:

• Known allergy to creatine or maltodextrin.
• History of adverse heat-related events (e.g., heat exhaustion, heat stroke, severe dehydration requiring medical intervention)
• Reports use of medications or supplements that may interfere with thermoregulation, heart rate, kidney injury or blood pressure responses during exercise (e.g. NSAID)