Purpose: Modern pentathlon is a multidisciplinary sport that involves exhaustive training which can cause tissue damage and metabolic changes. However, few studies have evaluated the metabolic changes that occur in pentathletes. Accordingly, we aimed to evaluate the metabolomic profile of pentathletes during a 3-week training period before competition using nuclear magnetic resonance. Methods: Blood samples from 6 members of a Brazilian modern pentathlon team were collected at the beginning (Pre1, Pre2, and Pre3) and end (Post1, Post2, and Post3) of each week. Low molecular-weight metabolite profiles were analyzed by nuclear magnetic resonance spectroscopy, and biochemical markers were assessed using enzyme-linked immunosorbent assays. Data were assessed using partial least-squares discriminant analysis and univariate statistical model. Results: Metabolic changes were observed between pre- and postdata of each week and over the 3 weeks before the competition in the partial least-squares discriminant analysis. Creatine kinase concentration increased in the first 2 weeks (P = .045 and P = .039), but there was no difference in the last week (P > .05). Lactate levels increased significantly after training in each week (P < .001). Cortisol levels at Post3 were different from all other time points (P < .05) and the concentrations of peroxides increased over the weeks (P < .05). Among all metabolites, sarcosine showed the greatest differences (P = .004) in the pretraining and posttraining periods of the 3 weeks. Conclusion: Serum analysis of athletes using nuclear magnetic resonance showed metabolic changes depending on the intensity of the training performed each week.