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Multiple sclerosis (MS) is an immune-mediated, neurological disease that results in physiological deconditioning with increasing disability. High-intensity interval training (HIIT) exercise has induced significant improvements in physiological conditioning in healthy and clinical populations and might be appropriate for persons with MS who have mobility disability. The feasibility and acute effects of HIIT using recumbent stepping in persons with MS who have mobility disability are relatively unknown.The physiological effects of single sessions of HIIT and continuous (CON), steady-state aerobic exercise using recumbent stepping were compared in 20 persons with MS with mobility disability (i.e., Expanded Disability Status Scale of 4.0–6.5). The HIIT bout included 10 cycles of 1-min intervals at the work rate associated with 90% peak aerobic capacity (V˙O2peak) followed by 1-min recovery intervals at 15 W, totaling 20 min in length. The CON bout consisted of 20 min at the work rate associated with 50% to 60% V˙O2peak. Physiological (i.e., power output, oxygen consumption, carbon dioxide expiration, respiratory exchange ratio, ventilation, HR, and core temperature) and perceptual (i.e., ratings of perceived exertion) measures were collected across the acute sessions.There were statistically significant condition–time interactions for all physiological measures and ratings of perceived exertion expressing differential patterns of change over time for HIIT versus CON (P < 0.05). The main effect of condition was significant for all physiological outcomes, except core temperature, with the HIIT condition inducing significantly higher values than CON (P < 0.05).High-intensity interval training exercise taxes the cardiorespiratory system significantly more than CON, yet without deleterious effects on core temperature in persons with MS. This has important implications for informing an evidence-based exercise prescription that is appropriate for improving physiological conditioning in persons with MS who have mobility disabilities.