To reduce the adverse consequences of exertion-related and acute intentional dehydration research has focused on monitoring hydration status. This investigation: 1) compared sensitivity of urine specific gravity (Usg), urine osmolality (Uosm) and a criterion measurement of hydration, plasma osmolality (Posm), at progressive stages of acute hypertonic dehydration and 2) using a medical decision model, determined whether Usg or Uosm accurately reflected hydra-tion status compared to Posm among 51 subjects tested throughout the day. Incremental changes in Posm were observed as subjects dehydrated by 5% of body weight and rehydrated while Usg and Uosm showed delayed dehydration-related changes. Using the medical decision model, sensitivity and specificity were not significant at selected cut-offs for Usg and Uosm. At the most accurate cut-off values, 1.015 and 1.020 for Usg and 700 mosm/kg and 800 mosm/kg for Uosm, only 65% of the athletes were correctly classified using Usg and 63% using Uosm. Posm, Usg, and Uosm appear sensitive to incremental changes in acute hypertonic dehydration, however, the misclassified outcomes for Usg and Uosm raise concerns. Research focused on elucidating the factors affecting accurate assessment of hydration status appears warranted.