PURPOSE: We investigated the effects of caffeine on the ammonia and amino acid metabolism of elite soccer players. METHODS: In this double-blind randomized study, athletes (n = 19) received 5 mg·kg caffeine or lactose (LEx, control) and performed 45 min of intermittent exercise followed by an intermittent recovery test (Yo-Yo IR2) until exhaustion. The caffeine-supplemented athletes were divided into two groups (CEx and SCEx) depending on their serum caffeine levels (<900% and >10,000%, respectively). Data were analyzed by ANOVA and Tukey post hoc test (P < 0.05 was considered to be statistically significant). RESULTS: Caffeine supplementation did not significantly affect the performance (LEx = 12.3 ± 0.3 km·h, 1449 ± 378 m; CEx = 12.2 ± 0.5 km·h, 1540 ± 630 m; SCEx = 12.3 ± 0.5 km·h, 1367 ± 330 m). Exercise changed the blood concentrations of several amino acids and increased the serum concentrations of ammonia, glucose, lactate, and insulin. The LEx group showed an exercise-induced increase in valine (∼29%), which was inhibited by caffeine. Higher serum caffeine levels abolished the exercise-induced increase (∼24%-27%) in glutamine but did not affect the exercise-induced increase in alanine (∼110%-160%) and glutamate (42%-61%). In response to exercise, the SCEx subjects did not exhibit an increase in uremia and showed a significantly lower increase in their serum arginine (15%), citrulline (16%), and ornithine (ND) concentrations. CONCLUSIONS: Our data suggest that caffeine might decrease systemic urea by decreasing the glutamine serum concentration, which decreases the transportation of ammonia to the liver and thus urea synthesis.