Low water intake is associated with long-term adverse health outcomes. Despite this, few studies had examined hydration physiology in the general population, where adequate intake, and not excessive loss, is the primary determinant of hydration. This study sought to evaluate the responsiveness of hydration biomarkers in urine and blood to acute changes in water intake. Key findings were that markers of urine concentration and urine volume change rapidly in response to a change in water intake, whereas plasma osmolality is maintained. Moreover, urine concentration varies as a function of the time of day, with nighttime and morning urine samples being more concentrated than afternoon samples. These results suggest that short, well-timed urine samples, such as those taken in the afternoon, may provide values comparable with 24-hour collections, which are more time-consuming and less convenient.