Jason Howitt

Aim Physical exercise triggers the secretion of small extracellular vesicles (sEVs) into the circulation in humans, enabling signalling crosstalk between tissues. Exercise‐derived EVs and their cargo have been proposed to mediate adaptations to exercise; however, our understanding of how exercise‐derived EV protein cargo is modulated by factors such as aerobic fitness and age of an individual is currently unknown. Here, we examined the circulating sEV proteome following aerobic exercise in healthy males of different ages and aerobic fitness to understand exercise‐induced EV response during the aging process. Methods Twenty‐eight healthy men completed a bout of 20‐min cycling exercise at 70% estimated VO 2peak . Small EVs were isolated from blood samples collected before and immediately after exercise, and then quantified using particle analysis and Western blotting. Small EV proteome was examined using quantitative proteomic analysis. Results We identified a significant increase in 13 proteins in small plasma EVs following moderate‐to‐vigorous intensity exercise. We observed distinct changes in sEV proteome after exercise in young, mature, unfit, and fit individuals, highlighting the impact of aerobic fitness and age on sEV protein secretion. Functional enrichment and pathway analysis identified that the majority of the significantly altered sEV proteins are associated with the innate immune system, including proteins known to be damage‐associated molecular patterns (DAMPs). Conclusion Together, our findings suggest that exercise‐evoked acute stress can positively challenge the innate immune system through the release of signalling molecules such as DAMPs in sEVs, proposing a novel EV‐based mechanism for moderate‐to‐vigorous intensity exercise in immune surveillance pathways.