Evaluating the relationship between step-rate and rate of oxygen uptake (VO2) may allow for practical physical activity assessment in patients with multiple sclerosis (MS) of differing disability levels.To examine whether the VO2 to step-rate relationship during over-ground walking differs across varying disability levels among patients with MS and to develop step-rate thresholds for moderate- and vigorous-intensity physical activity.Adults with MS (N=58; age: 51±9years; 48 women) completed one over-ground walking trial at comfortable speed, one at 0.22m·s−1 slower, and one at 0.22m·s−1 faster. Each trial lasted 6min. VO2 was measured with portable spirometry and steps with hand-tally. Disability status was classified as mild, moderate, or severe based on Expanded Disability Status Scale scores.Multi-level regression indicated that step-rate, disability status, and height significantly predicted VO2 (p<0.05). Based on this model, we developed step-rate thresholds for activity intensity that vary by disability status and height. A separate regression without height allowed for development of step-rate thresholds that vary only by disability status.The VO2 during over-ground walking differs among ambulatory patients with MS based on disability level and height, yielding different step-rate thresholds for physical activity intensity.