Metabolomic profiling of intensity-dependent responses to acute exercise in healthy humans

Exercise confers numerous health benefits, yet the intensity-specific impact of acute exercise on systemic metabolism remains incompletely characterized. This study aimed to characterize serum metabolomic responses to different treadmill-based exercise intensities using liquid chromatography–mass spectrometry (LC–MS) and to assess associations between metabolite signatures and exercise capacity indicators. We conducted a randomized controlled trial (ClinicalTrials.gov Identifier: NCT04830059). Fifty-eight healthy, inactive university students were randomized to high-intensity interval training (HIIT, n = 20), moderate-intensity continuous training (MICT, n = 19), or light-intensity training (LIT, n = 19). Serum samples were collected at rest and immediately post-exercise. An untargeted LC–MS metabolomics approach was used to quantify alterations in 480 serum metabolites across the three protocols. Acute HIIT, MICT, and LIT significantly altered 204, 198, and 60 serum metabolites, respectively, with a shared core of 37 metabolites. Metabolic perturbations were clearly intensity-dependent: HIIT elicited the most profound shifts, characterized by rapid accumulation of glycolytic, TCA-cycle, and antioxidant-associated metabolites. MICT induced broad activation of glycolysis, the TCA cycle, and fatty acid oxidation, whereas LIT caused milder changes primarily in glycolysis and bile acid signaling. Across all intensities, strong positive correlations between lactate and TCA-cycle intermediates underscored the tight coupling between glycolytic flux and mitochondrial respiration. Baseline VO2max and HRmax showed distinct metabolite association profiles, with VO2max positively related to amino acid–derived and redox-related metabolites, whereas HRmax was linked to organic acid, bile acid, and dipeptide species. This parallel-group metabolomics trial demonstrates that a single bout of treadmill exercise elicits intensity-dependent, yet partly overlapping, metabolic programs spanning central carbon, amino acid, lipid, and bile acid pathways. By integrating lactate-centered correlations with VO2max- and HRmax-related metabolite signatures, our findings outline a molecular framework linking acute exercise intensities metabolism to cardiorespiratory fitness and provide a basis for refining intensity-specific exercise prescriptions. The study was registered on ClinicalTrials.gov (Identifier: NCT04830059). This randomized controlled trial maps distinct serum metabolomic signatures for high-intensity interval training (HIIT), moderate-intensity continuous training (MICT), and light-intensity training (LIT). A core metabolic scaffold involving central carbon pathways is shared across all three intensities. HIIT drives rapid energy flux, MICT promotes fat oxidation, and LIT primarily modulates bile acid signalling. Lactate acts as a central hub connecting glycolysis, mitochondrial oxidation, and lipid remodelling. VO2max correlates with antioxidant and amino acid–related metabolites, whereas HRmax links to organic acids, bile acids, and dipeptides.