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 SOD1G93A ALS mouse model, we conducted a longitudinal study to identify the early signs of defect to pinpoint the potential mechanism for diaphragm dysfunction. Oxygen consumption of mitochondria isolated from diaphragm muscles was impaired at high energy demands in hSOD1G93A mice (p=0.0014) at 12 weeks old, compared with that in WT mice, and continued to worsen as disease progression ensued. This mitochondrial functional decline precedes the dismantlement of neuromuscular junction (NMJ) structure, which appeared at 18 weeks of age (p=0.0016, with 57±4% fully innervated in hSOD1G93A diaphragm vs. 78± 2.4% in WT), while peak diaphragmatic contractile force measured using ex vivo muscle contraction was preserved until 22 weeks of age (hSOD1G93A 143±12.2mN vs WT: 231±8.1mN). The late decline in peak contractile force in diaphragm is not surprising in the context of preserved breathing function in ALS mice. In-vivo transdiaphragmatic pressure measurements confirmed the impairment of respiratory function at 22 weeks old during maximal chemoreceptor stimulation and total tracheal occlusion. Further, diaphragm revealed no significant changes in myosin heavy chain isoforms at 12 weeks old, consistent with the lack of denervation at 12 weeks old, but shifted to slower isoforms (Type IIB to Type IIA/X) at 22 weeks of age. These results together indicate that mitochondrial defect in diaphragm is potentially a primary player for respiratory dysfunction in ALS and resolving mitochondrial dysfunction may delay the onset of respiratory failure at late stages of disease progression.