Postpartum Exercise Enhances the Proteomic Cargo of Human Breast Milk Small Extracellular Vesicles

Introduction and Objective: Early-life nutrition influences lifelong metabolic health. Breast milk contains small extracellular vesicles (sEVs) that carry bioactive metabolic signals. Although exercise remodels sEV proteomic cargo in plasma, its effect on breast milk sEV composition is unknown. This study investigates if a single bout of moderate-intensity aerobic exercise alters the proteomic composition of breast milk sEVs. Methods: Eleven healthy lactating women completed two days of standardized meals prior to study visits, including an exercise trial and a time-matched sedentary visit. Baseline breast milk samples were collected on the second pump of the day after 1hr of supervised rest. Participants then remained sedentary or walked on an incline for 30 minutes at 70% of VO2max. At 30 minutes post-exercise (time-matched to 90 minutes post-baseline pump on the sedentary visit) and 120 minutes post-exercise breast milk was collected for isolation of sEVs using size-exclusion chromatography and ultracentrifugation. Proteomic cargo was analyzed by mass spectrometry. Results: Exercise induced robust remodeling of breast milk sEV proteomic cargo, reflected by 44% and 71% of identified proteins significantly elevated (FDR < 0.05) at 30 and 120 minutes post-exercise, respectively. No changes were observed on the sedentary day. Proteomic analysis identified elevated extracellular vesicle markers (based on Vesiclepedia) indicating greater sEV content. Further analysis revealed enrichment of translation pathways post-exercise, suggesting delivery of proteo-static machinery relevant to early-life development. Conclusion: These findings demonstrate that moderate-intensity aerobic exercise increases breast milk sEV proteomic cargo, highlighting an unexplored link between maternal physical activity and infant metabolic development. Disclosure: V. Parmar: None. S.S. Loona: None. D. Gupta: None. K. Sevits: None. K. Klaus: None. C.J. Heppelmann: None. L.M. Szymanski: None. A. Egan: None. M. Pataky: None.