Little is known about the impact of habitual fluid intake on physiology. Specifically, biomarkers of hydration status and body water regulation have not been adequately explored in adults who consume different fluid volumes in everyday conditions, without prolonged exercise or environmental exposure. The purpose of the present study was to compare adults with habitually different fluid intakes with respect to biomarkers implicated in the assessment of hydration status, the regulation of total body water and the risk of kidney pathologies. In the present cross-sectional study, seventy-one adults (thirty-two men, thirty-nine women, age 25–40 years) were classified according to daily fluid intake: thirty-nine low drinkers (LD; ≤ 1·2 litres/d) and thirty-two high drinkers (HD; 2–4 litres/d). During four consecutive days, urinary parameters (first morning urine (FMU) on day 1 and subsequent 24 h urine (24hU) collections), blood parameters, and food and beverage intake were assessed. ANOVA and non-parametric comparisons revealed significant differences between the LD and HD groups in 24hU volume (1·0 (se0·1) v. 2·4 (se 0·1) litres), specific gravity (median 1·023 v. 1·010), osmolality (767 (se 27) v. 371 (se 33) mOsm/kg) and colour (3·1 (se 0·2) v. 1·8 (se 0·2)). Similarly, in the FMU, the LD group produced a smaller amount of more concentrated urine. Plasma cortisol, creatinine and arginine vasopressin concentrations were significantly higher among the LD. Plasma osmolality was similar between the groups, suggesting physiological adaptations to preserve plasma osmolality despite low fluid intake. The long-term impact of adaptations to preserve plasma osmolality must be examined, particularly in the context of renal health.