Introduction: Multiple sclerosis (MS) is a chronic autoimmune disease that leads to inflammation, demyelination, and neurodegeneration in the central nervous system, causing damage to both white and grey matter. This disruption affects motor and sensory pathways, leading to a wide range of physical and cognitive impairments. Fatigue is the most commonly reported symptom in MS, significantly impacting daily life and exercise adherence. The underlying causes of MS-related fatigue are complex, involving central mechanisms such as impaired signal propagation and axonal loss. Understanding motor unit behaviour, particularly during fatigue, offers valuable insights into central nervous system dysfunction in MS and could improve treatment approaches for MS-related fatigue. Methods: 11 people with MS (mean age = 44 ± 9) and 11 age- and gender-matched controls participated in this study. Participants attended a 2 hour in-person testing session at the Neural Basis of Movement Laboratory at Griffith University on the Gold Coast. During testing, participants completed two fatigue questionnaires (the Fatigue Severity Scale and Modified Fatigue Impact Scale), four clinical tests (6-minute walk test, timed sit to stand test, stair climb test and wall squat hold), and a series of isometric knee extension contractions. During the isometric knee extension tasks, high density electromyography (HD-EMG) data was recorded from the vastus lateralis muscle of the participant's dominant leg. Participants were required to complete voluntary knee extensions at 30%, 50% and 70% of their maximal voluntary contraction. These contractions were completed in an unfatigued state and then again following a task designed to induce fatigue to the muscle. Results: Compared to healthy controls, participants with MS exhibited reduced maximal knee extension (~10-50%) torque, significantly poorer outcomes on all clinical tests, and reported higher levels of fatigue in the questionnaires. Motor unit analysis from HD-EMG data revealed that discharge rate increased with increasing contraction intensity across both the control and MS groups, and decreased after the introduction of motor fatigue. Spike count was significantly lower for the MS group compared to the control group in the presence of motor fatigue. Spike variability was significantly higher for the MS group (~200%) compared to the control group in the presence of motor fatigue. Notably, individuals with MS showed increased motor unit coherence in the 0-5 Hz delta band, indicative of volitional force control, as well as in the 5-15 Hz alpha and 15-35 Hz beta bands, associated with afferent input regulation and descending motor commands, respectively. Furthermore, following correlative analysis, the combination of clinical tests used in this study were able to explain 23.57% of the coefficient of variation of the interspike interval and 32.76% of mean motor unit discharge characteristics in people with MS. Discussion: In this study, people with MS demonstrated reduced motor unit discharge rates, reduced torque steadiness, and higher exertion perception during submaximal and fatiguing contractions compared to healthy controls. Intra-muscular coherence analysis revealed altered synaptic input contributions, with MS showing coherence in the 5-15 Hz alpha and 15-35 Hz beta bands. While clinical tests had little impact on baseline discharge rates, this study found that while the selected clinical tests have little relationship to the variance in baseline motor unit discharge rate, there is some predictive value when examining fatigued discharge rates and baseline coefficient of variation of the interspike interval.