Amyotrophic Lateral Sclerosis (ALS) is a debilitating motor neuron degenerative disease characterized by progressive motor neuron death. eventually leading to limb paralysis, respiratory failure, and death within 2-5 years of diagnosis. As majority of patients with ALS ultimately succumb to respiratory failure, understanding the mechanisms for the decline in respiratory function during disease progression is essential for the development of effective therapeutic strategies to prolong survival. Here, using the well-characterized hSOD1G93A ALS mouse model, we conducted a longitudinal study, and discovered that oxygen consumption of mitochondria isolated from diaphragm muscles was impaired at high energy demands in hSOD1G93A mice (p=0.0014) at 12 weeks old, pressure (PDI) measurements confirmed the impairment of respiratory function at 22 weeks old during total tracheal occlusion (p=0.0194). In our lab, hSOD1G93A mice were crossed with a new doxycycline (DOX)-inducible (TetOn System) dominant-negative mitochondrial calcium uniporter (dnMCU) model that selectively reduces Ca2+ uptake in skeletal muscle (hSOD1G93A/SM-dnMCU). We found that it improves muscle contractile function at late stages of the disease. In conjunction with a 10-week exercise program, it further attenuates disease progression at advanced and late stages of the disease. At 22 weeks (late stage), PDI significantly improved (p=0.01 and p=0.018) in the hSOD1G93A/SM-dnMCU exercise trained cohort compared to both untrained and hSOD1G93A /dnMCU WT littermates. Peak diaphragmatic contractile force measured using ex vivo muscle contraction also improved at 22 weeks of age in the hSOD1G93A/SM-dnMCU exercise trained vs hSOD1G93A /dnMCU WT littermates. These results together indicate that mitochondrial defect in diaphragm is potentially a primary player for respiratory dysfunction in AL and synergistic effects of attenuating mitochondrial Ca2+ uptake in SM and a novel concurrent exercise strategy to improve mitochondrial health and delay the debilitating effects of ALS on respiratory dysfunction.