Biomarkers of hydration change in response to acute dehydration; however, their responsiveness to changes in fluid intake volume, without exercise or heat exposure, has not been adequately described. Moreover, patterns of circadian variation in hydration biomarkers have not been established. The study aims were to (1) assess the response of hydration biomarkers to changes in daily water intake; and (2) evaluate circadian variation in urinary and salivary biomarkers. Fifty-two adults (24.8 ± 3.1 years; 22.3 ± 1.6 kg/m(2); 79 % female), grouped based on habitual fluid intake (low drinkers, n = 30, <1.2 L/day; high drinkers, n = 22, >2.0 L/day), completed a 5-day inpatient crossover trial. On days 1 and 2, low drinkers received 1.0 L/day of water while high drinkers received 2.5 L/day. On days 3 through 5, intake was reversed between groups. Plasma and saliva osmolality were assessed daily at predetermined times, and all urine produced over 24 h was collected in timed intervals. ANOVA with intake (1.0 vs. 2.5 L/day), day, and time revealed that (1) urine concentration (osmolality, specific gravity, color) and volume, but not plasma nor saliva osmolality, responded to changes in water intake; (2) urinary hydration biomarkers and saliva osmolality vary as a function of the time of day; and (3) urine osmolality measured in samples collected during the afternoon most closely reflects the corresponding 24 h value. Overall, urinary hydration biomarkers are responsive to changes in water intake, and stabilize within 24 h of modifying intake volume. Moreover, short afternoon urine collections may be able to replace 24 h collections for more convenience in hydration assessment.