Limited data exists regarding the relationship between dietary protein intake and fluid balance. Current trends towards increased consumption of dietary protein in athletes necessitate an evaluation of the effects of high protein intake on hydration status. Hydration status was evaluated in six endurance runners (mean ± SEM, 20 ± 0.6y, 70 ± 1.2kg, 9 ± 0.5% body fat, 180 ± 5cm, VO2peak 71 ± 1.9ml/kg/min) who participated in a crossover design experiment following random assignment to either a high (3.6 g/kg, HP) or moderate (1.8 g/kg, MP) protein diet. The diets were isocaloric, and the macronutrient composition for carbohydrate, protein, and fat were 55%, 15%, 30%, and 40%, 30%, 30% for MP and HP, respectively. Fluid balance indices included body mass, daily fluid intake, perceived thirst, urine color, urine specific gravity, and plasma osmolality. With the exception of plasma blood urea nitrogen (BUN) and osmolality, all measurements were made biweekly throughout the 4-week dietary interventions. Plasma BUN was determined at 3 weeks and plasma osmolality was determined at three weeks and following a 75 min run at submaximal intensity (70% VO2peak). Body mass remained unchanged throughout the study and no differences were noted in daily fluid intake or thirst between the two dietary interventions. However, urine specific gravity was significantly higher for HP vs. MP (1.021 ± 0.004 vs. 1.019 ± 0.004, p < 0.05) and plasma BUN was greater for HP vs. MP (7.5 ± 0.6 vs. 4.4 ± 0.4 mmol/L, p < 0.05). In addition, plasma osmolality was also significantly higher, both pre- and post- exercise with HP vs. MP (Pre-287 ± 4 vs. 281 ± 4, p < 0.05, and Post-295 ± 13 to 287 ± 3, p < 0.050). These data suggest that protein intake did not influence fluid balance. However, the exceptional elevation in plasma BUN (normal range, 2.5–6.4 mmol/L) coupled with higher plasma osmolality and urine specific gravity indicates a greater renal solute load with HP due to excretion of nitrogenous byproducts. Therefore, prolonged consumption of high protein diets by endurance athletes may necessitate increased water intake and continuous monitoring of hydration status. Supported by National Cattlemen's Beef Association