This study aims to characterize the relationship between increased protein intake and hydration indexes. Five men participated in a 12-week, randomized, crossover, controlled diet intervention study. Subjects consumed eucaloric diets containing 3.6 (high protein), 1.8 (moderate protein), and 0.8 (low protein) g/kg/day of protein for 4 weeks each. Energy intakes were based on requirements established relative to resting energy expenditure and activity at baseline. Assessments included blood urea nitrogen, plasma osmolality, urine-specific gravity, and estimates of fluid balance. Repeated-measures analyses of variance and paired t tests were used to determine effects of treatment and time. Fluid intake and fluid balance were unaffected. Blood urea nitrogen was higher for high protein vs low protein and vs moderate protein, and urine-specific gravity was higher for high protein vs moderate protein. Baseline plasma osmolality was greater for high protein vs low protein and vs moderate protein. The effect of increasing dietary protein on fluid status was minimal.