Extracellular vesicles (EVs) are small lipid bilayer-delimited particles that are secreted by all cells, playing a central role in cellular communication. EVs are released from skeletal muscle during exercise, but the effects of contractile activity on skeletal muscle-derived EVs (Skm-EVs) are poorly understood due to the challenges in distinguishing Skm-EVs derived from exercising muscle in vivo. Using tunable resistive pulse sensing (TRPS), we previously demonstrated that chronic contractile activity (CCA) increased the secretion of Skm-EVs from C2C12 myotubes, while their size and zeta potential remained unchanged. Here, we aimed to determine whether similar results could be obtained using an alternative method of EV characterization, dynamic light scattering (DLS). C2C12 myoblasts were differentiated into myotubes, and electrically paced (3h/day x 4days @14V, C-PACE EM, IonOptix) to mimic chronic exercise in vitro. EVs were isolated from conditioned media of control and stimulated myotubes using differential ultracentrifugation, and characterized based on size and zeta potential. The mean size of EVs from chronically stimulated myotubes (CCA-EVs, 132 nm) was 26% smaller than control (CON-EVs, 178 nm). Size distribution analysis revealed that CCA-EVs were enriched in small EVs (100-150 nm), while CON-EVs were largely composed of 200-250 nm sized vesicles. Additionally, zeta potential was 27% lower in CCA-EVs compared to CON-EVs. Our data indicate that the effect of CCA on facilitating the release of smaller, more stable EVs, is a robust finding, reproducible by multiple methods of EV characterization. Future studies investigating the mechanisms by which CCA influences Skm-EV biogenesis and secretion are warranted.