Physiological and functional characterization for high‐throughput optogenetic skeletal muscle exercise assays

Exercise promotes human mobility by tuning the function of skeletal muscle, and recent studies highlight exercise's broader impacts on human health via muscle's paracrine and endocrine roles beyond force generation. In vitro models of tissue engineered skeletal muscle enable precise investigation of adaptation to exercise, with emerging approaches for optogenetic muscle stimulation providing a less invasive alternative to traditional techniques for electrical stimulation. In this study, we present a high‐throughput muscle culture and optical exercise protocol for scalable in vitro exercise studies. First, we characterize optical rheobase for 2D muscle monolayers, finding that optical intensities as low as 5 μW mm −2 can trigger functional contraction. We then leverage RNA sequencing to map changes in muscle gene expression in response to various optical exercise regimens, highlighting how changing stimulation parameters impact myogenic and broader physiological and pathological transcriptional responses. Our platform and results establish a practical foundation for high‐throughput in vitro exercise studies of skeletal muscle.