Research assessing exercise-induced hypohydration on running performance in a temperate environment is scarce. Given the weight-bearing nature of running, the negative effects of hypohydration might be offset by the weight-loss associated with a negative fluid balance. Therefore, this study investigated the effect of exercise-induced hypohydration on running performance in temperate conditions. Seventeen intermittent games players (age 22 +/- 1 y; VO2peak 52.5 +/- 4.1 mL.kg(-1).min(-1)) completed preliminary and familiarisation trials, and two experimental trials consisting of 12 blocks of 6 min of running (65% VO2peak; preload) with 1 min passive rest in-between, followed by a 3 km time trial (TT). During the preload, subjects consumed minimal fluid (60 mL) to induce hypohydration (HYP) or water to replace 95% sweat losses (1622 +/- 343 mL; EUH). Body mass loss (EUH -0.5 +/- 0.3%; HYP -2.2 +/- 0.4%; P < 0.001), and other changes indicative of hypohydration, including increased serum osmolality, heart rate, thirst sensation, and decreased plasma volume (P = 0.022), were apparent in HYP by the end of the preload. TT performance was similar to 6% slower in HYP (EUH 900 +/- 87 s; HYP 955 +/- 110 s; P < 0.001). Exercise-induced hypohydration of similar to 2% body mass impaired 3 km running TT performance in a temperate environment.