Fatigue is one of the most limiting symptoms in people with multiple sclerosis (pwMS) and can be subdivided into trait and state fatigue. Activity-induced state fatigue describes the temporary decline in motor and/or cognitive performance (motor and cognitive performance fatigability, respectively) and/or the increase in the perception of fatigue (perceived fatigability) in response to motor or cognitive tasks. To the best of our knowledge, the effects of a 6-min walk test (6MWT), which was often used to assess motor performance fatigability in pwMS, on motor-cognitive dual-task performance (i.e., walking + arithmetic task) and prefrontal cortex (PFC) hemodynamics are not well-known. This is of importance, since daily activities are often performed as multitasks and a worse dual-task walking performance is associated with an increased risk of falling. Consequently, we investigated the effect of a fast 6MWT (comfort velocity + 15%) performed on a treadmill on motor-cognitive performance fatigability (spatio-temporal gait parameters/accuracy during the arithmetic task) and perceived fatigability measures (rating of perceived exhaustion; RPE) as well as PFC hemodynamics recorded during dual-task walking in pwMS and healthy controls (HCs). Twenty pwMS (48.3 ± 9.0 years; 13 females/7 males; expanded disability status scale 2.7 ± 1.0, first diagnosis 13.8 ± 8.8 years) and 24 HC with similar age and sex (48.6 ± 7.9 years; 17 females/7 males) were included. Only cognitive performance fatigability (increased error rate) during dual-task walking was found after the fast 6MWT on the treadmill in pwMS. However, the changes in gait parameters did not indicate motor performance fatigability, although both the groups reported perceived fatigability (increased RPE) after the fast 6MWT. Moreover, no change in the PFC activation was detected in both groups. Our results suggest that the intensity and/or duration of the fast 6MWT was not sufficient to induce motor performance fatigability in pwMS. These factors should be addressed by future studies on this topic, which should also consider further parameters, e.g., muscular oxygenation and/or myoelectrical activity, to verify that exercise intensity and/or duration was appropriate to induce motor performance fatigability in pwMS.Clinical Trial RegisterDRKS00021057.