Motor neuron disease (MND) is a progressive neurodegenerative disease characterised by motor neuron death and the loss of voluntary muscle control. The irreversible loss of motor neurons and their connections with skeletal muscle leads to muscle atrophy, weakness and paralysis. While the fundamental cause for MND remains unknown, recent evidence suggests that impairments in skeletal muscle metabolism may contribute to disease progression. In order to characterise the pathophysiological nature of skeletal muscle bioenergetics in MND, we developed a method that allows for the real-time assessment of metabolic flux in intact extensor digitorum longus (EDL) muscle fibres from the SOD1G93A mouse model of MND and age-matched wildtype (WT) littermates at pre-defined disease stages. In spite of the remarkably decreased muscle size, we find that EDL muscle fibres from SOD1G93A mice exhibit basal respiration rates that are comparable to WT controls, indicating that basal energy demands do not decline with worsening muscle pathology. By assessing the flexibility and maximal capacity of EDL fibres to oxidise glucose or long-chain fatty acid as energy substrates, we demonstrate preferential use of fatty acid as an energy source and enhanced mitochondrial oxidation capacity in EDL fibres of SOD1G93A mice when compared to WT controls. Our data are in line with previous studies that report impaired glucose metabolism in MND, and suggest that metabolic adaptions towards fatty acid oxidation may occur to maintain the energy demands of skeletal muscle throughout disease progression. By assessing skeletal muscle bioenergetic profiles in situ, our study provides insight into metabolic pathways that may be modulated to potentially improve muscle symptoms in MND.