PROJECT ABSTRACT Anterior cruciate ligament (ACL) injury is a common orthopedic injury that results in persistent quadriceps weakness that drives poor patient outcomes and increases the risk of post-traumatic osteoarthritis (PTOA). Despite extensive rehabilitation, and regardless of whether the ACL is surgically reconstructed, only 1 in 5 patients regain acceptable levels of quadriceps strength. As ACL injury causes an immediate shutdown of neural signaling, neuromuscular electrical stimulation (NMES) is commonly prescribed to activate inhibited motoneurons, thereby improving quadriceps activation and permitting strength recovery. Although NMES is widely used, our own meta-analysis and other data show that the clinical success is inconsistent, likely due to tremendous diversity in stimulation intensities and how soon treatments are initiated after injury. Research that improves our understanding of the optimal intensity and timing of this treatment to maximize its effectiveness would be immediately impactful. Our new breakthrough data highlight an important and overlooked relationship between the loss of neural activation and mitochondrial dysfunction as important contributors to muscle deficits after ACL injury. Neural inhibition, i.e., the loss of action potentials, disrupts post-synaptic calcium signaling that triggers mitochondria to produce excess reactive oxygen species known to severely compromise muscle health. These data reinforce our rationale to optimize NMES as this therapy can directly depolarize inhibited motoneurons in the absence of volitional neural activation to maintain the electrical properties of muscle necessary for contraction and mitochondrial health. To understand how to optimize the delivery, we developed a non-invasive rat model that faithfully replicates the clinical injury. The objective of this proposal is to use this preclinical model to test specific NMES treatment parameters and the underlying mechanisms of action. Aim 1 will define the intensity and time of treatment initiation that maximizes positive muscle outcomes using a custom-built rodent dynamometer that will translate the intensity of stimulus and strength outcomes to the human condition. Aim 2 will test the ability of optimized NMES to be protective of knee joint health by reducing risk factors for PTOA after ACL injury. Aim 3 will determine the clinical importance between the loss of neural activation and mitochondrial dysfunction, and explore whether future clinical applications should consider the concurrent use of mitochondrial-targeted antioxidant therapies after ACL injury. As NMES is part of the standard of care for ACL injury, this work will provide fundamental knowledge to guide clinical practice.