Hydration and Post-exercise Hypotension

The intervention for the current study is hydration status (see below). Participants will complete a total of 3 visits to the laboratory, 1 familiarisation and 2 experimental visits. Each participant will complete all 3 visits (no separate arms), and at no point will the participant or researchers be blinded to the requirements of that visit. The first visit is a familiarisation and screening session, designed to determine the eligibility of a participant, answer any questions the participant may have, gain consent from the participant, and familiarise the participant with all study procedures. The following 2 visits will be completed in a counterbalanced order and are designed to determine the effect of hydration status on the post-exercise hypotension. For visit 2 and 3 participants will complete 30 minutes of fixed intensity cycling exercise, and then complete 30 minutes of standing, during which physiological measures will be taken (e.g. blood pressure and heart rate). For simplicity the visits are ordered to indicate the different requirements and are labelled as follows: Visit 2 - Hypohydration Visit 3 - Euhydration (serves as active control) For visits 2 and 3, the primary outcome of interest is the change in blood pressure from pre-intervention (e.g. resting baseline) to post-intervention (e.g. following cycling exercise) for each participant (a within-subject design). All 3 study visits will occur face-to-face, in a temperature-controlled laboratory, and be led by a senior researcher with extensive research experience and 20 years of exercise physiology research experience. The first visit will take ~1 hour and the remaining 2 visits will take ~approximately 1.5 hours. Each visit will be separated by at least 5 days. The information collected will include the following: Demographics (Visit 1) • Date of birth, age, sex • Height and weight • Participant contact details • Current exercise regimen Medical history (Visit 1) • Other medical conditions and medications • Smoking history: smoking status (ex, current, never) • Females: self-reported pregnancy status and menstrual cycle phase All visits • Adverse events • Serious adverse events Cycling exercise A functional threshold power (FTP) test will be used to estimate the highest cycling power output that can be sustained for 60 minutes. The FTP test will be conducted during the familiarisation session (visit 1) using a IC6 indoor cycle (Life Fitness, Rosemont, Illinois, USA). Briefly, a warm-up consisting of cycling for 5 minutes at a cadence of 50-60 rpm at a self-selected cadence to achieve a rate of perceived exertion (RPE - see below) of 5/10. Following the warm-up a ramp-test will be completed, which includes stepwise increases in resistance (5 levels every minute whilst maintaining a cadence of 85-90 rpm), until the participant reports an RPE of 10/10. The resistance will then be reduced to achieve an RPE of 5/10 whilst maintaining a cadence of 40-50 rpm for a total of 7 minutes of active recovery. Upon completion of the active recovery period the participant will begin the FTP test. The resistance will be increased to the level achieved at the completion of the ramp test and the participant will then attempt to maintain a cadence of 85-90 rpm for 5 minutes. The resistance can be decreased if desired. The power output for the fixed intensity cycling test in visits 2 and 3 will be 80% of the power output from this FTP test. The fixed-intensity cycling exercise (visits 2 and 3) will be performed on a stationary IC6 indoor cycle . Participants will cycle at 80% of their estimated FTP for 30 minutes, preceded by a 5-minute warm-up at 50% FTP and followed by a 3-minute cool-down at 40% FTP. During visits 2 and 3 post-exercise, participants will stand for 30 minutes, with BP taken at 5-minute intervals for a total of 30 minutes. A safety threshold of a 30 mmHg decrease in systolic BP from the pre-exercise seated baseline (within visit) will be used. If this threshold is exceeded, or if the participant develops any symptoms associated with fainting, including light-headedness, nausea, blurry vision, weakness, or shortness of breath, the visit will be terminated and the participant will be assisted to a seated position. Rate of perceived exertion (RPE) RPE is a scale used to assess how hard participants feel they are working. This study will use a modified Borg CR10 RPE scale where 0 represents no exertion and 10 represents maximal effort. RPE will be measured during the FTP test described above, and at 5 minute intervals during the fixed intensity cycling exercise. Visit 1 – familiarisation (1 hour) Prior to the familiarisation, a call will be made to potential participants to assess initial eligibility. If deemed eligible in the initial screening, a familiarisation session will be scheduled at a time convenient for the participant. At the time of scheduling, participants will be asked to abstain from strenuous exercise and alcohol for 12 hours, and caffeine (e.g. coffee, tea or energy drinks) for 4 hours prior to arriving at the laboratory. Written informed consent will be obtained. If participants are eligible: • Height and body weight will be recorded. • The participant will be fitted to the cycle ergometer, with handlebar and seat settings recorded for consistency across visits. • Instruction and correction on proper cycling technique will be provided. • An FTP test will be conducted to provide an estimate of their FTP. • Testing of workload for subsequent visits will be performed. Visits 2 and 3 will involve identical procedures, with the only difference being hydration status. Participants will be asked to abstain from strenuous exercise and alcohol for 12 hours, and caffeine (e.g. coffee, tea, or energy drinks) for 4 hours, prior to arriving at the laboratory. Participants will also be asked to complete a 12-hour diet recall sheet and refrain from food with vasoactive components (e.g. nitrates) as outlined on the diet recall sheet provided (further detail provided below). Both visits will be completed at the same time of day (±1 hour) starting no later than 9am. For female participants visits 2 and 3 will be repeated during the same phase of the menstrual cycle, to control for the potentially confounding effect of ovarian hormones on arterial function. Self-reported menstrual cycle information will be requested by a female member of the study team during the scheduling of visits 2 and 3. Due to the within-subject design, visits 2 and 3 can be completed during any menstrual cycle phase (e.g. early follicular or luteal). However, the timing will be kept consistent across visits 2 and 3, such that all visits will occur in the same menstrual cycle phase e.g. visits 2 and 3 both occur during the luteal phase. If the participant is using oral contraception or an intra-uterine device, if applicable we will ask for self-reported phase of use (e.g. placebo-pill phase) such that the visits can also be scheduled in the same phase. Visits 2 and 3 Hydration status assessment To assess hydration status, participants will be asked to provide a urine sample for assessment of urine specific gravity (USG) upon arrival to the laboratory. • Hypohydration (HYP) condition defined as USG less than1.020 • Euhydration (EUH) condition defined as USG greater than 1.020 • Water intake will be restricted following provision of the USG sample in both visits. Water can then be consumed ad libitum once the final blood pressure measure has been recorded (30 minutes post exercise). Visit 2 – Hypohydration visit (1 hour 30 minutes) Participants will be instructed that they may consume a bolus of water up to 1L, 18 hours before the visit. After this, participants will be asked to refrain from consuming water or other liquids (e.g. coffee, milk, tea, energy drinks, soups/broths) for the 18-hour period before the visit. Water consumption will be restricted until the visit is complete. If the USG of the sample provided by the participant is 1.018, another urine sample will be obtained after 30 minutes. If USG remains 1.023, then the visit will be rescheduled. Visit 3 - Euhydration visit (1 hour 30 minutes - serve as active control) For the EUH visit, participants will be instructed to maintain EUH during the 12h prior to exercise. They will be asked to keep their normal hydration habits while ensuring they consume a minimum of 500 ml the night before the visit and the same volume again for the morning of the visit. Once the participant has provided a urine sample, water consumption will be restricted until the visit is complete. If the USG is >1.020 but 1.020, then the visit will be rescheduled. If the initial USG returns a value of >1.023, then the visit will be rescheduled. Participant information and education Cycling technique Cycle ergometer set-up will be facilitated by the researchers in visit 1. Fainting symptoms Participants will be informed of the symptoms of fainting in visit 1, and reminded in all visits before and after exercise. Diet recall Participants will be asked to repeat their diet for the 12 hours prior to visit 2, and 3. As visits 3 and 4 are counterbalanced, the participant will be asked to keep a diet diary for the 12 hours prior to whichever visit occurs first, and then repeat this diet prior to remaining visits. Participants will be provided with a food recall diary at visit 1 and a list of vasoactive foods that should be avoided prior to each visit. Adherence to the diet requirements will be self-reported. The diet diary will be returned at the final study visit. In said diet recall sheet instructions regarding the consumption of water and other foods will be provided Restrictions prior to all visits: • Strenuous exercise and alcohol for 12 hours, and caffeine (e.g. coffee, tea, or energy drinks) for 4 hours, prior to arriving at the laboratory. Foods to avoid 12 hours prior to your laboratory visit: • Beetroot and beetroot juice • Dark chocolate • Berries including blackcurrants and grapes • Nutritional supplements containing high nitrate or anthocyanin content