Background: This study aimed to examine muscle fatigue in lower leg muscles in of people with multiple sclerosis and healthy controls, and whether muscle fatigue coincided with potential changes in gait. Methods: In this case-control study, people with multiple sclerosis (n = 8; 3male; mean age (SD) = 49.7 (9.6) yr) and age-matched healthy controls (n = 10; 4male; mean age (SD) = 47.4 (8.7) yr) walked on a treadmill for 12-min at self-paced speed. Muscle fatigue was indirectly quantified by a decrease in median frequency and increase in root mean square of surface electromyographic recordings of lower leg muscles. Walking speed, ankle push-off power and net ankle work were calculated from marker positions and force plate data using inverse dynamic calculations. Results: People with multiple sclerosis showed larger decreases in median frequency of soleus (most affected leg: p = 0.003; least affected leg: p = 0.009) and larger increases in root mean square of soleus (most and least affected leg: p = 0.037), gastrocnemius medialis (most affected leg: p = 0.003; least affected leg: p = 0.005) and lateralis (most and least affected leg: p < 0.001) compared to controls. Walking speed (p = 0.001), ankle push-off power (most affected leg: p = 0.018; least affected leg: p = 0.001) and net work around the ankle (most affected leg: p = 0.046; least affected leg: p = 0.001) were lower in people with multiple sclerosis compared to controls, but increased in both groups. Interpretation: The results yield preliminary evidence that soleus muscles of people with multiple sclerosis fatigue during prolonged walking. Changes in electromyography of gastrocnemius muscles could however be related to muscle fatigue, changes in gait or a combination.