Regular exercise evokes numerous systemic benefits, including enhanced mitochondrial function, protection against cardiovascular diseases, and potential anti-tumorigenic effects, among many others. Strong evidence suggests that the systemic adaptative effects of exercise are potentiated in part by the release of myokines during exercise from skeletal muscle. However, the precise molecular mechanisms driving the adaptations of exercise have yet to be fully elucidated. Beyond myokines, extracellular vesicles (EVs), key mediators of intercellular communication, are also released from skeletal muscle during exercise. EVs are small, lipid bilayer-enclosed structures that carry several biomolecules from host cells to recipient cells, thereby modulating the function of target cells. Given the established role of skeletal muscle in exercise-induced responses and the central role of EVs in cell signaling, we hypothesized that skeletal muscle-derived EVs (Skm-EVs) may contribute to the systemic benefits associated with exercise. To investigate this, we employed chronic contractile activity (CCA) on differentiated C2C12 myotubes to mimic exercise in vitro and examined, for the first time, the effects of Skm-EVs on recipient cells. We hypothesized that Skm-EVs derived post-CCA (or CCA-EVs) would mediate the pro-metabolic and anti-tumorigenic effects associated with chronic exercise. In our first study, we observed that CCA led to an increased release of Skm-EVs, which subsequently enhanced mitochondrial biogenesis in C2C12 myoblasts, indicating a potential mechanism for the pro-metabolic benefits of exercise. In the second study, we demonstrated that CCA-EVs increased apoptosis and senescence in Lewis Lung Carcinoma cells, suggesting a role in the anti-tumorigenic effects of exercise. Importantly, in both studies, pre-treatment of CCA-EVs with proteinase K, with or without Triton X-100, reduced their effect on recipient cells, highlighting the significance of the membrane-associated EV protein cargo in mediating these effects. Building on these findings, we conducted a third study to analyze the proteomic content of Skm-EVs and identified potential protein candidates that might be involved in mediating the pro-metabolic and anti-tumorigenic effects of CCA-EVs in the first two studies. Collectively, these findings advance our understanding of the role of Skm-EVs in mediating the systemic benefits of exercise, potentially providing a mechanistic basis for the development of exercise-based therapeutic strategies.