This study examined the effect of repeated familiarisation to hypohydration on hypohydrated exercise performance. After familiarisation with the exercise protocol, 10 recreationally active males completed a euhydrated (EU-pre) and hypohydrated (HYPO-pre) trial, which involved a 45-min steady state run at 75% peak oxygen uptake (45SS) followed by a 5-km time trial (TT). Euhydration and hypohydration were induced by manipulating fluid intake in the 24-h pre-exercise and during the 45SS. Subjects then completed 4 habituation sessions that involved replication of the HYPO-pre trial, except they completed 60 min of running at 75% peak oxygen uptake and no TT. Subjects then replicated the euhydrated (EU-post) and hypohydrated (HYPO-post) trials. Body mass loss pre-TT was 0.2 (0.2)% (EU-pre), 2.4 (0.3)% (HYPO-pre), 0.1 (0.1)% (EU-post), and 2.4 (0.3)% (HYPO-post). TT performance was 5.8 (2.4)% slower during the HYPO-pre trial (1459 (250) s) than during the EU-pre trial (1381 (237) s) (p < 0.01), but only 1.2 (1.6)% slower during the HYPO-post trial (1381 (200) s) than during the EU-post trial (1366 (211) s) (p = 0.064). TT performance was not different between EU-pre and EU-post trials, but was 5.1 (2.3)% faster during the HYPO-post trial than the HYPO-pre trial (p < 0.01). Heart rate was greater during HYPO trials than EU trials (p < 0.001), whilst rating of perceived exertion (RPE) response was similar to TT time and was lower in the HYPO-post trial than the HYPO-pre trial (p < 0.01). In conclusion, hypohydration impaired 5-km running performance in subjects unfamiliar with the hypohydration protocol, but 4 familiarisation sessions designed to habituate subjects with the hypohydration protocol attenuated the performance decrement, seemingly via an attenuation of RPE during hypohydrated exercise.