The aim of this study was to determine the intraocular pressure response to differing levels of dehydration. Seven males participated in 90 min of treadmill walking (5 km h(-1) and 1% grade) in both temperate (22 °C) and hot (43 °C) conditions. At baseline and 30 min intervals intraocular pressure, nude body mass, body temperature and heart rate were recorded. Statistically significant interactions (p < 0.05) were observed for intraocular pressure (hot condition: baseline 17.0 ± 2.9, 30 min 15.6 ± 3.5, 60 min 14.5 ± 3.7 and 90 min 13.6 ± 2.9 mmHg; temperate condition: baseline 16.8 ± 2.7, 30 min 16.5 ± 2.6, 60 min 15.8 ± 2.5 and 90 min 15.7 ± 1.8 mmHg) and body mass loss (hot condition: 30 min -1.07 ± 0.35, 60 min -2.17 ± 0.55 and 90 min -3.13 ± 0.74%; temperate condition: 30 min -0.15 ± 0.11, 60 min -0.47 ± 0.18 and 90 min -0.78 ± 0.25%). Significant linear regressions (p < 0.05) were observed for intraocular pressure and body mass loss (adjusted r(2) = 0.24) and intraocular pressure change and body mass loss (adjusted r(2) = 0.51). In conclusion, intraocular pressure was progressively reduced during a period of exercise causing dehydration, but remained relatively stable when hydration was maintained. The present study revealed a moderate relationship between dehydration (body mass loss) and intraocular pressure change.