To determine if inhibition of MPT, mROS, or caspase-3 prevents atrophy in a single muscle fiber model of disuse atrophy, single isolated FDB myofibers were kept in culture conditions and treated with DMSO (vehicle control),1 µM TR002 (MPT-inhibitor), 20 µM Ac-ATS010-KE (caspase-3 inhibitor), or 5 µM mitoTEMPO (mROS-inhibitor) for 4 days, and myofiber diameter was assessed pre- and post-treatment. (A) Representative images of isolated mouse FDB fibers following 4-days of treatment with DMSO, TR002, Ac-ATS010, or mitoTEMPO. Scale bars represent 100 µm. Biological replicates (n = 2) are identified by data points of different colors within a given treatment, while technical replicates share the same color data point. (B) Inhibition of MPT, caspase-3, and mROS all prevented change in myofiber diameter over the 4-day period, while the vehicle control group demonstrated a substantial decline in size. This indicates that inhibition of MPT, caspase-3, or mROS prevent atrophy in a culture model of skeletal muscle disuse. * p < 0.05.