Dehydration impairs endurance exercise performance, especially in the heat. However, it is unclear how exercise performance is affected by improved hydration status in underhydrated individuals. Therefore, this study examined the impact of a one-week hydration intervention on endurance running performance. We hypothesized better hydration would lead to a faster 2-mile running time. Eighty-nine healthy and moderately fit individuals completed a 2-mile run as fast as possible before (PRE) and after (POST) a 1-week hydration intervention. Performance was incentivized by providing prize money based on ranked average times. First-morning urine samples were collected on the day of each run. If PRE urine specific gravity was >1.020, the subjects were instructed to increase their water intake for one week before the second run. The remaining subjects were instructed to maintain their water intake. All subjects were provided with a filter water bottle. Of the 89 subjects that completed both runs, 44 (18% female, age: 21±2 y, weight: 73.3±12.5 kg, height: 1.76±0.10 m, body mass index: 23.6±3.2 kg/m2) were included in the analysis. Subjects were classified as improved hydration (IMP) if UOsm decreased between PRE and POST by ≥100 mmol/kg and POST was <800 mmol/kg. Subjects were classified as diminished hydration (DIM) if UOsm increased between PRE and POST by ≥100 mmol/kg and POST was >500 mmol/kg. The subjects were removed from the analysis if they met any of the following three conditions: a) had a small UOsm change (<100 mmol/kg) between PRE and POST, b) lowered UOSM but it was still >800 mmol/kg, and c) increased UOsm but it was still <500 mmol/kg. The 2-mile runs took place in Phoenix, Arizona, during the morning (0700 hours) with a maximum ambient temperature of 22±2℃, relative humidity of 37±10%, and WBGT of 16±2℃. UOsm significantly decreased for IMP from PRE: 791±197 to POST: 419±136 mmol/kg (p<0.05) and increased for DIM from PRE: 532±283 to POST: 799±235 mmol/kg (p<0.05). The 2-mile run time significantly improved in IMP from PRE: 13.5±1.8 to POST: 13.3±1.8 min (-11.4±25.8 sec, P=0.03), but did not change in DIM from PRE: 14.2±2.7 to POST: 14.3±2.6 min (+4.8±19.9 sec, P>0.05). The results suggest that improving hydration in underhydrated subjects via one week of increased water intake may improve 2-mile running performance in moderately fit individuals. This study was funded by a grant from the Arizona State University’s Global Sport Institute. Brita donated the filter water bottles. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.