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INTRODUCTION: HDL particle functionality is influenced by its structure, including lipid composition. However, the effects of exercise training on the HDL lipidome and its relationship with HDL-related traits are largely unknown.OBJECTIVE: To investigate the HDL lipidome of 154 adults before and after 20 weeks of endurance exercise training in the HERITAGE Family Study.METHODS: The HDL-sized plasma fraction was isolated utilizing FPLC-SEC, followed by untargeted lipidomic analysis using LC/MS. A total of 11 HDL lipid classes were derived from the 341 identified known lipid species. Exercise response of the HDL lipidome and its associations with HDL-related traits were examined, with significance set to FDR < 0.05.RESULTS: The abundance of 42 HDL lipid species at baseline and 43 at post-training were significantly different between males and females. Exercise training did not significantly alter the abundance of any HDL lipid class, although HDL phosphatidylethanolamine trended (FDR = 0.05) towards an increase. Two species of HDL diglycerides significantly decreased in the total sample. Sex-specific nominal (p < 0.05) changes in individual HDL lipid species included primarily HDL diglyceride and triglyceride species decreasing in males only, while HDL phosphatidylethanolamine species mostly increasing in females only. Higher abundance of HDL surface lipids was associated with larger size and cholesterol content of HDL particles before and in response to exercise training.CONCLUSION: Our analysis indicates that endurance exercise may have a limited impact on the HDL lipidome in healthy adults. However, the HDL lipidome differed across sex groups, which needs further investigation to identify potential mechanisms underlying the sex differences.