Blood serum osmolality (S (OSM)) is the gold standard to assess body fluid balance. Urine specific gravity (U (SG)) is also a body fluid balance index but it is not invasive. However, U (SG) capability to detect the minimal level of dehydration that affects athletic performance (i.e., 2 %) remains untested. We collected urine and blood samples in eighteen euhydrated trained athletes in the morning and that evening while dehydrating by 1, 2, and 3 % of body mass by cycling (60 % VO₂peak) in the heat (32 °C, 46 % rh, 2.5 m s(-1) air flow). At 9:00 pm, subjects left the laboratory and went to bed after ingesting 0.7 ± 0.2 L of a sports drink. The next morning, subjects awoke 3 % hypohydrated, and blood and urine samples were collected and test terminated. We found that 2 % dehydration increased S (OSM) and U (SG) above exercise-baseline values (P < 0.05). The next morning, S (OSM) and U (SG) remained elevated compared to the first morning while euhydrated (287 ± 5 vs. 282 ± 3 mOsmol kg(-1) H(2)O and 1.028 ± 0.003 vs. 1.017 ± 0.005, respectively, P < 0.05). However, when comparing 3 % dehydration (end of exercise) to 3 % hypohydration (next morning), U (SG) increased (1.025 ± 0.003 to 1.028 ± 0.003; P < 0.05) while S (OSM) decreased (295 ± 5 to 287 ± 5 mOsmol kg(-1) H(2)O; P < 0.05). In summary, during exercise-induced dehydration, U (SG) is as sensitive as S (OSM) to detect low levels of dehydration (i.e., 2 %). Both indices maintain the ability to detect a 3 % overnight hypohydration although S (OSM) approaches euhydration values, while U (SG) remains a superior index to detect hypohydration.