Approach to methods: To our knowledge, the effect of hydration status on glycemia and appetite has never been causally investigated in healthy adults. The aim of this study design was to dehydrate participants, then either rehydrate, or continue dehydrating them, in a randomized crossover design. We then measured the glycemic and metabolic responses in the rehydrated (RE) and hypohydrated (HYPO) states. The methods were chosen based on several factors, including participant comfort, reliability, ecological validity, and theory regarding the effect of different methods of hypohydration on the mechanisms of interest (e.g. intravenous hypohydration, fluid restriction, exercise-induced hypohydration). Further, we aimed to utilise methods to assess the glycemic response that are commonly used in clinical and research settings (e.g. the use of an oral glucose tolerance test, used in both clinical practice and research, rather than hyperinsulinemic euglycemic clamp, used primarily in specific research settings).   Several methods have previously been utilised to induce hypohydration. In a study of 15 participants (n = 10 men) with type 1 diabetes1, hypohydration was induced via fluid restriction (750 mL/24 h) and both oral and intravenous diuretic drugs. Participants were weighed every six hours until = 3 % of their baseline body mass had been lost. Another study in nine men with type 2 diabetes2 hypohydration was achieved via three days of fluid restriction (1 L·d-1 on days 2 and 3 prior to the trial, and 0.5 L·d-1 on the day preceding the trial). Both of these studies in patients with diabetes showed a higher glycemic response when hypohydrated compared to euhydrated.   In a study of 10 healthy male participants3, hyperosmolality was induced via complete fluid restriction and intravenous infusion of hypertonic NaCl over 17 h. Whilst hyperosmolality was achieved, body mass remained stable and urinary output did not notably change, suggesting whole body hydration status was not altered. Although intravenous infusion was an effective method of inducing a hyperosmotic state, it lacks ecological validity, limiting the inferences to specific clinical or research settings. Further, these methods are more likely to hone in on specific mechanisms, such as increased arginine vasopressin (AVP) secretion. In our pilot study 4 of five healthy participants (n = 4 men), hypohydration was induced via 45 min dehydration in a sauna (55-85°C) followed by fluid restriction (200 mL) over ~14 h, achieving 0.6-1.6 % body mass loss. This method of hypohydration is likely to be more representative of whole body water losses, rather than compartmental, as per Keller et al.3.   Based on these pilot data4, achieving 1-2 % hypohydration based on body mass was hypothesized to achieve a clear increase in the glycemic response during an oral glucose tolerance test. Although we did not aim for a specific level of hypohydration, the methods in the current project were therefore designed to stay within this limit as 1-2 % hypohydration has been reported as achievable in the general population5.   The methods from this pilot study were deemed an adequate compromise of controlling dehydration, and having external validity, and were therefore utilised in the current project. The final procedure we used (60 min in a heat tent) was based on feedback from participants as well as findings from the pilot study regarding participant comfort and estimated weight loss. Thus, in order to achieve the initial hypohydration, participants were overnight fluid restricted, then put in a heat tent wearing a sweat suit for one hour. Whilst the use of a heat tent reduces the ecological validity of the study, it was chosen as a method to help standardise the procedure and minimise participant discomfort from extended fluid restriction. Though being in a sauna increases AVP6, participants were dehydrated in this manner in both trial arms, thus controlling for any residual confounding effects on AVP during testing (approximately 20-24 hours post-heat tent).   In terms of the fluid prescriptions post-heat tent, participants were instructed on how much to drink per hour during RE to ensure even water intake distribution across the day until 2200 h. All other fluids were prohibited, but during RE participants could drink more water than prescribed. The use of total (rather than lean) body mass for the HYPO procedure was to increase participant comfort by allowing slightly more fluid without meaningfully altering their hydration state. References: 1. Burge MR, Garcia N, Qualls CR, Schade DS. Differential effects of fasting and dehydration in the pathogenesis of diabetic ketoacidosis. Metabolism: clinical and experimental 2001; 50(2): 171-177. doi: 10.1053/meta.2001.20194   2. Johnson EC, Bardis CN, Jansen LT, Adams JD, Kirkland TW, Kavouras SA. Reduced water intake deteriorates glucose regulation in patients with type 2 diabetes. Nutrition research 2017; 43(2): 25-32. doi: 10.1016/j.nutres.2017.05.004   3. Keller U, Szinnai G, Bilz S, Berneis K. Effects of changes in hydration on protein, glucose and lipid metabolism in man: impact on health. European journal of clinical nutrition 2003; 57 Suppl 2: S69-74. doi: 10.1038/sj.ejcn.1601904   4. Carroll HA, Johnson L, Betts JA. Effect of hydration status on glycemic control: A pilot study. In: American College of Sports Medicine. Boston, MA., 2016.   5. Armstrong LE. Hydration Biomarkers During Daily Life. Nutrition Today 2012; 47: S3-S6. doi: 10.1097/NT.0b013e31826266cf   6. Bussien JP, Gaillard RC, Nussberger J, Waeber B, Hofbauer KG, Turnill D et al. Haemodynamic role of vasopressin released during Finnish sauna. Acta endocrinologica 1986; 112(2): 166-171. doi: 10.1530/acta.0.1120166