Objectives: To analyze the effect in the blood metabolome of trail running, a demanding sport that takes place in the natural environment, places considerable strain on both muscles and joints. While metabolic responses to aerobic exercise have been analyzed in-depth, few studies have focused on trail running. Design: Observational study to analyze changes in 35 different metabolites - representative of aerobic exercise- induced by a simulated 21-km trail race with an uphill gradient of 1400 m. Methods: We performed a semiquantitative metabolomics study consisting of capillary blood microsampling and targeted screening with liquid chromatography and mass spectrometry to analyze, in 33 licensed athletes, changes concerning 35 metabolites. Results: We observed significant changes in many metabolites, including increased acetyl-carnitine and taurine concentrations (false discovery rate–corrected paired t-test P value 1.63 × 10–13, and P value 5.021 × 10-12, re- spectively) and decreased carnitine and proline concentrations (P value 6.33 × 10–10, and P value 1.21 × 10–9, re- spectively). Metabolic responses to trail running were largely independent of sex but were influenced by the level of training, with runners with a higher level showing resistance to exercise-induced changes in taurine, 1-methyl histidine, acetyl-carnitine, and hypoxanthine concentrations. Performance (measured as race time) was inversely correlated with changes in specific metabolites (including taurine, serotonin, and hypoxanthine) and directly correlated with increases in glutathione. Conclusions: Our findings demonstrate the usefulness of metabolomics studies for analyzing exercise-induced physiological changes and show individual differences associated with the level of training and performance.