Non‐Targeted Analysis of Extracellular Vesicle‐Enriched Plasma Proteome Between Early and Late Rugby Playing Career

ABSTRACT Purpose Rugby players experience high‐impact collisions, potentially increasing their risk of neurodegenerative conditions. This study investigates whether the plasma proteome of extracellular vesicles (EV) provides biomarkers to indicate differential risk associated with a rugby career. Experimental Design Twenty‐four males were recruited: eight academy rugby players (18 ± 1 years), eight professional rugby players (33 ± 5 years; >10‐year career), and eight CrossFit athletes (32 ± 5 years; no history of collision‐related injuries). EV were enriched from plasma using strong‐anion exchange magnetic microparticles and digested proteins were analyzed by LC‐MS/MS for label‐free quantitation. Results A total of 449 proteins were identified (false discovery rate <1%). Statistical analysis on 403 proteins quantified in at least n = 3 participants in each group highlighted 52 significant ( p < 0.05, q < 0.01) differences, including 44 proteins that had abundance profiles unique to professional rugby players. Eight proteins which were depleted and three proteins which were elevated have previously recognized roles in neurodegenerative processes. Conclusions and Clinical Relevance Proteins associated with neuroprotection were specifically depleted in the plasma EV proteome of long‐serving professional rugby players. The proteins highlighted in professional rugby players could be used to develop biomarker panels for predicting at‐risk athletes or for guiding treatment interventions. Summary Repetitive high‐impact collisions experienced by rugby players may predispose them to neurodegenerative conditions, yet the biological processes underpinning this risk remain poorly understood. This study investigates whether the proteome of plasma extracellular vesicles (EV) could serve as early, minimally invasive biomarkers of neurodegenerative risk in athletes exposed to repeated head impacts. By comparing the EV proteome of professional rugby players, younger academy athletes, and non‐collision sport controls, we identified specific proteins with known neuroprotective roles that were depleted in long‐serving rugby professionals. These alterations suggest systemic biological changes related to prolonged exposure to collisions. Our findings provide novel insight by highlighting the potential of EV‐based proteomic profiling as a tool for early detection and monitoring of neurodegeneration‐related processes in at‐risk athletic populations. This approach could ultimately inform strategies for risk stratification, early intervention, and tailored clinical monitoring in collision sport athletes.