Electrical pulse stimulation (EPS) is used to study exercise-related adaptations in muscle cells in vitro. We compared the effects of two EPS protocols: 1. intermittent high-frequency or 2. continuous low-frequency stimulation, on glucose and fatty acid metabolism, muscle fiber-type markers, mitochondrial proteins, myokines and extracellular vesicles (EV) release. Both EPS protocols enhanced post-exercise glycogen synthesis, while an increase in oxidative capacity was observed only after the intermittent high-frequency stimulation. Both EPS protocols increased GDF11 protein and release of EVs, which were characterized by transmission electron microscopy, nanoparticle tracking analysis and proteomics.