Objectives/Goals: Our team has developed a 24-well donor-derived skeletal muscle microphysiological system (MPS) to study signaling pathways associated with a variety of muscle diseases. 3D muscle will be utilized to evaluate pharmacologic interventions for these muscle conditions to improve both muscle mass and function. Methods/Study Population: In this study, muscle MPS were formed from healthy young female and male subjects. 3D muscle underwent a 21-day differentiation with an electrical stimulation (e-stim) regimen twice daily beginning on Day 14. Functional assessments in permeabilized fibers of both sexes included isometric and isotonic calcium-induced contractions, allowing for the characterization of force-pCa (-log[Ca2+]), force-velocity and force-power relationships. Samples from Day 17 and Day 21 will be assessed for pro-growth protein signaling via western blotting and a subset of samples will be analyzed by histology and microscopy for fiber type and size. Finally, culture media pre- and post-terminal e-stim on Day 21 will be collected for extracellular vesicle (EV) isolation and EVs will be assessed by standard proteomics analysis. Results/Anticipated Results: Permeabilized fibers from both sexes reproduced the well-established sigmoidal force-pCa and the curvilinear force-velocity and force-power relationships reported in native striated muscle. Maximum specific force and force-pCa relationship were not different between sexes. Isotonic contractile measurements revealed that these male and female fibers also exhibit similar force-velocity and force-power relationships. We anticipate that 3D muscles from day 17 compared to day 21 will exhibit higher levels of pro-growth protein signaling due to e-stim application and no differences in fiber type or size between sexes. Additionally, we expect that EV quantity will depend upon 3D muscle maturity and presence of e-stim. Discussion/Significance of Impact: This study demonstrates the similarities of functional characteristics and exercise (or e-stim) adaptation between native human skeletal muscle and 3D bioengineered skeletal muscle. Ultimately, this data further validates the muscle MPS system to study muscle diseases and to enhance the translation of therapeutics to clinical settings.