The delineation of exercise biochemistry by utilizing metabolic fingerprinting has become an established strategy. We present a combined RP-UPLC-MS and H-1 NMR strategy, supplemented by photometric assays, to monitor the response of the human urinary metabolome to short maximal exercise. Seventeen male volunteers performed two identical sprint sessions on separate days, consisting of three 80 m maximal runs. Using univariate and multivariate analyses, we followed the fluctuation of 37 metabolites at 1, 1.5, and 2 h postexercise. 2-Hydroxyisovalerate, 2-hydroxybutyrate, 2-oxoisocaproate, 3-methyl-2-oxovalerate, 3-hydroxyisobutyrate, 2-oxoisovalerate, 3-hydroxybutyrate, 2-hydroxyisobutyrate, alanine, pyruvate, and fumarate increased 1 h postexercise and then returned toward baseline. Lactate and acetate were higher than baseline at 1 and 1.5 h. Hypoxanthine and inosine remained above baseline throughout the postexercise period. Urate decreased at 1 h and increased at 1.5 h before returning to baseline. Valine, isoleucine, succinate, citrate, trimethylamine, trimethylamine N-oxide, tyrosine, and formate decreased at 1 h and/or 1.5 h postexercise and then returned to baseline. Creatinine gradually decreased over the sampling period. Glycine, 4-aminohippurate, and hippurate remained below baseline throughout the postexercise period. Our findings show that even one-half minute of maximal exercise elicited major perturbations in human metabolism, several of which persisted for at least 2 h.