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