### Summary **BACKGROUND**: Cardiorespiratory fitness, commonly measured as peak oxygen consumption (peak VO₂, is a strong predictor of cardiovascular health. Circulating lipids have been linked to exercise responses and metabolic adaptation, but the bioactive lipid species associated with objectively measured aerobic capacity and their dynamic regulation during exercise remain incompletely defined. **METHODS**: We performed directed, nontargeted liquid chromatography-mass spectrometry profiling of circulating bioactive lipids in adults undergoing clinically indicated cardiopulmonary exercise testing, relating resting lipid levels and acute exercise-induced lipid changes to peak VO₂. We then examined fitness-associated lipid species across complementary models of physiological stress, including marathon running, chronic voluntary wheel running in mice, and the postprandial Burmese python model. **RESULTS**: Among 1896 detected lipid species in the primary human cohort, 47 were associated with peak VO₂ after false-discovery rate correction, including 16 metabolites with known molecular identities. Higher levels of several eicosanoids and free fatty acids were associated with greater peak VO₂, including docosahexaenoic acid, arachidic acid, and 12,13-dihydroxy-9Z-octadecenoic acid. In paired rest and peak exercise samples, 37 of 47 fitness-associated lipid species changed acutely with exercise. Among metabolites with known identities, exercise-induced changes in 12,13-dihydroxy-9Z-octadecenoic acid, 8-hydroxyicosa-9,11,14-trienoic acid, 11,12-epoxy-5Z,8Z,14Z-eicosatrienoic acid, and 8,9-dihydroxy-5Z,11Z,14Z-eicosatrienoic acid were associated with peak VO₂. In marathon runners, 43 of 47 fitness-associated lipid species changed after prolonged endurance exercise. Exploratory animal analyses demonstrated overlapping lipid regulation in chronic voluntary wheel-running mice and in the postprandial Burmese python model. **CONCLUSIONS**: Circulating bioactive lipids, particularly eicosanoids and free fatty acids, are associated with human cardiorespiratory fitness and are dynamically regulated during exercise. Overlapping lipid responses across complementary human and animal models identify candidate lipid pathways that may merit future mechanistic study.