The separation and identification of circulating small extracellular vesicles from endurance-trained, strength-trained and recreationally active men.

Small extracellular vesicles (EV) are membrane-encapsulated particles that carry bioactive cargoes, are released by all cell types and are present in all human biofluids. Changes in EV profiles and abundance occur in response to acute exercise, but this study investigated whether individuals with divergent histories of exercise training (recreationally active controls - CON; endurance-trained - END; strength-trained - STR) presented with varied abundances of small EVs in resting samples and whether the abundance of small EVs differed within each group across two measurement days. Participants (n = 38, all male; CON n = 12, END n = 13, STR n = 13) arrived at the lab on two separate occasions in a rested, overnight fasted state, with standardisation of time of day of sampling, recent dietary intake, time since last meal and time since last exercise training session (∼40 h). Whole blood samples were collected and separated into plasma from which small EVs were separated using size exclusion chromatography and identified in accordance with the Minimal Information For Studies of Extracellular Vesicles (MISEV) guidelines. No differences in the abundance of small EVs were observed within or between groups across multiple methods of small EV identification (nanoparticle tracking analysis, flow cytometry, immunoblot of specific EV markers). Targeted metabolomics of the small EV preparations identified 96 metabolites that were associated with the structure and function of small EVs, with no statistically significant differences in concentrations observed across groups. The results of the current study suggest that the abundance and metabolomic profile of small EVs derived from men with divergent histories of exercise training are similar to those in resting blood samples. KEY POINTS: Extracellular vesicles (EV) are membrane-encapsulated particles that are present in circulation and carry bioactive materials as 'cargo'. The abundance and profile of small EVs are responsive to acute exercise, but little is known about the relationship between small EVs and exercise training. This study examined the abundance, and a targeted metabolomic profile, of small EVs separated from the blood of endurance athletes, strength athletes and recreationally active controls at rest (∼40 h after the most recent exercise session) on two separate but identical lab visits. No differences were observed in the abundance or metabolomic profile of small EV preparations between the groups or between the lab visits within each group. Further research should determine whether the bioactive cargoes (e.g. RNA, protein and additional metabolites) carried within EVs are altered in individuals with divergent histories of exercise training or in response to exercise training interventions.