Individual Responses to Physical Activity: Study of the Relation Between Different Types of Biomolecular Predictors and the Main Parameters of Aerobic and Resistance Exercise. Focusing on Circulating Extracellular Vesicles as Biomarkers of Individual Exercise Response and Trainability, Moving Toward Personalized Monitoring of Training Adaptations in Athletes and Tactical Populations

Physical exercise induces complex systemic adaptations involving coordinated molecular responses across tissues. Extracellular vesicles (EVs) have emerged as key mediators of intercellular communication, transferring proteins, lipids, and nucleic acids that influence physiological adaptation. Their responsiveness to exercise stress positions them as promising biomarkers of individual trainability, yet the regulation of their release and cargo across distinct exercise regimens remains unclear. This PhD project explored how the release, molecular composition, and functional properties of EVs are modulated by different exercise forms. Three studies investigated EVs from saliva, serum, and plasma to capture acute and chronic adaptations to physical stress. Salivary EVs were examined in professional soccer players during preseason training to evaluate early molecular responses to exercise. Nanoparticle Tracking Analysis confirmed expected size distribution and concentration, while transmission electron microscopy verified vesicle morphology. Flow cytometry analysis identified ExoBrite-positive vesicles expressing canonical tetraspanins CD9, CD63, and CD81, confirming their exosomal nature. Dot blot quantification revealed a transient rise in the stress-associated chaperone HSP60 normalized to CD63 at 15 hours post-exercise, suggesting an early, short-lived enrichment of stress-related cargo. These results support saliva as a practical and noninvasive source for monitoring acute EV-mediated stress responses in athletes. Serum EVs were analyzed from ultramarathon runners before and after a 200 km race to assess systemic responses to prolonged endurance exertion. EVs isolated by Size Exclusion Chromatography displayed stable CD63 expression but significant increases in CD9, HSP60, and the muscle-specific marker CAV3, reflecting selective modulation of EV composition under sustained metabolic and mechanical load. The detection of neural adhesion molecule L1CAM suggested broader inter-tissue communication involving the nervous system. Correlations between these EV markers and classical indicators of muscle damage, including CPK, LDH, and myoglobin, reinforced their potential as integrated indicators of exercise-induced physiological stress. Plasma EVs were characterized in U.S. Marines over six weeks of multi-stressor military training to assess cumulative adaptation. EV size, concentration, and canonical surface markers remained stable, while HSP60 displayed a near-significant increase at mid-training, indicative of transient mitochondrial stress. Proteomic profiling revealed modulation of corticosteroid-binding globulin (SERPINA6), apolipoprotein E (APOE), and versican (VCAN), implicating endocrine regulation, lipid metabolism, and extracellular matrix remodeling. Correlations between EV protein changes and measures of resilience and body mass were linked to pathways governing vesicle-mediated transport, IGF1 signaling, and metabolic adaptation, reflecting coordinated physiological and psychological responses to sustained stress. Collectively, these studies demonstrate that EVs carry distinct molecular signatures reflective of exercise-induced stress, recovery, and adaptation. By integrating findings across biological fluids and exercise models, this work supports circulating EVs as multidimensional, accessible biomarkers for personalized monitoring of training adaptation and resilience in both athletic and tactical populations.