People with multiple sclerosis (PwMS) tend to be less physically active than the general population. Limited physical activity increases fatigue, possibly affecting other functions such as balance. Treadmill training is a promising method to ameliorate these symptoms. The aim of this study was to assess the effect of treadmill training on fatigue and balance. Thirty PwMS were recruited; the mean age was 47.6 (SD 9.2). The median EDSS score was 5.5 (range 3–6.5). Individuals were randomized into a control group receiving 12 sessions of conventional therapy and an experimental group receiving conventional therapy including 15 min of treadmill training. Rate of perceived exertion (RPE), heart rate (HR), the Fatigue Severity Scale, and the Berg Balance Scale were assessed before and after rehabilitation. Despite a low HR (107 beats/min), the RPE score was high (15.8) at baseline assessment. In the experimental group, RPE decreased significantly to 12.8 (P=0.04) after training. Treadmill training also had a positive effect on HR, but no changes in the Fatigue Severity Scale and balance were observed. In conclusion, PwMS showed a high level of exertion before treatment. Treadmill training was effective in reducing the level of perceived fatigue, with no impact on balance.