Fatigue is often reported in stroke patients. However, it is still unclear if fatigue in stroke patients is more prominent, more frequent or more typical than in patients with multiple sclerosis (MS) and if the pathophysiology differs between these two populations. The purpose of this study was to compare motor fatigue and fatigue induced changes in kinematic gait parameters between stroke patients, MS patients and healthy persons. Gait parameters at the beginning and end of a treadmill walking test were assessed in 10 stroke patients, 40 MS patients and 20 healthy subjects. The recently developed Fatigue index Kliniken Schmieder (FKS) based on change of the movement’s attractor and its variability was used to measure motor fatigue. Six stroke patients had a pathological FKS. The FKS (indicating the level of motor fatigue) in stroke patients was similar compared to MS patients. Stroke patients had smaller step length, step height and greater step width, circumduction with the right and left leg as well as greater sway compared to the other groups at the beginning and at the end of test. A severe walking impairment in stroke patients does not necessarily cause a pathological FKS indicating motor fatigue. Moreover, the FKS can be used as a measure of motor fatigue in stroke and MS and may also be applicable to other diseases.