This study investigated (a)differences between males and females for changes in serum, tear, and urine osmolality, hematocrit, and urine specific gravity following acute passive dehydration and (b)assessed the reliability of these biomarkers separately for each sex. Fifteen males (age: 26.3 ± 3.5years, body mass: 76 ± 7kg) and 15 females (age: 28.8 ± 6.4years, body mass: 63 ± 7kg) completed a sauna protocol twice (5-28days apart), aiming for 4% body mass loss (BML). Urine, blood, and tear markers were collected pre- and postdehydration, and change scores were calculated. Male BML was significantly greater than that of females in Trial 1 (3.53% ± 0.55% vs. 2.53% ± 0.43%, p < .001) and Trial 2 (3.36% ± 0.66% vs. 2.53% ± 0.44%, p = .01). Despite significant differences in BML, change in hematocrit was the only change marker that displayed a significant difference in Trial 1 (males: 3% ± 1%, females: 2% ± 1%, p = .004) and Trial 2 (males: 3% ± 1%, females: 1% ± 1%, p = .008). Regression analysis showed a significant effect for sex (male) predicting change in hematocrit (β = 0.8, p = .032) and change in serum osmolality (β = -3.3, p = .005) when controlling for BML but not for urinary or tear measures. The intraclass correlation coefficients for females (ICC 2, 1) were highest for change in urine specific gravity (ICC = .62, p = .006) and lowest for change in tear osmolarity (ICC = -.14, p = .689), whereas for males, it was posthematocrit (ICC = .65, p = .003) and post tear osmolarity (ICC = .18, p = .256). Generally, biomarkers showed lower test-retest reliability in males compared with females but, overall, were classified as poor-moderate in both sexes. These findings suggest that the response and reliability of hydration biomarkers are sex specific and highlight the importance of accounting for BML differences.