Metabolomics is a comprehensive method for metabolite assessment that involves measuring the overall metabolic signature of biological samples. We used this approach to investigate biochemical changes due to acute and chronic physical exercise. Twenty-two women using identical oral contraceptives were segregated into an untrained (n = 10) or trained (n = 12) group depending on their physical training background. The subjects performed a short-term intensive exercise test (Wingate anaerobic test). Urine specimens were collected before and 30 min after each test. The samples were analyzed by 1H NMR spectroscopy, and multivariate statistical techniques were utilized to process the data. Higher creatinine excretion was observed in trained compared with untrained women. In both groups, the excretion of lactate, pyruvate, alanine, b-hydroxybutyrate, and hypoxanthine increased similarly after the completion of the Wingate test. The excretion of acetate increased to a lesser extent in trained than in untrained subjects. These metabolic changes may be related to the redox status and the oxidative stress of muscular cells. In conclusion, metabolomic investigations in the fields of pharmacology and toxicology and in clinical studies should consider intense physical activity as an important biological factor influencing the outcome of experiments. Moreover, metabolomics is a promising tool for profiling athletes' urine in order to gain insight into physiologic states and to clarify the changes induced by short-term intense physical exercise.