Background: Patients with multiple sclerosis (pwMS) often experience a decline in motor function and performance during prolonged walking, which potentially is associated with reduced ankle push-off power and might be alleviated through structured exercise. The objectives of this pilot study were to assess ankle push-off power and walking performance in pwMS and healthy controls, and the preliminary effectiveness of a sequential exercise program (resistance training followed by walking-specific endurance training) on ankle push-off power and walking performance. Methods: PwMS (N = 10) with self-reported reduced walking performance and healthy controls (N = 10; at baseline only) underwent 3D gait analysis during a self-paced 12-minute walking test to assess walking performance prior to and following a sequential exercise program. Secondary testing paradigms comprised isometric muscle testing (triceps surae), cardiopulmonary exercise testing and self-report measures. Findings: PwMS had a shorter 12-minute walking distance, and lower peak ankle push-off power (most-affected leg) in comparison to healthy controls. There was no minute-to-minute decline in walking performance. The 8-week resistance training significantly improved walking distance. In parallel, higher peak and speed-normalized ankle push-off power were found in the less-affected side. No additional changes were found following the walking-specific endurance training phase. Interpretation: There was no walking-related motor fatigue found during a self-paced 12-minute walking test despite reduced ankle push-off power, and self-reported walking problems. Preliminary effects suggest a positive effect of resistance training on walking performance, potentially associated with increases in ankle push-off power, interestingly, in the less-affected leg. The added effect of the walking-specific endurance training remains unclear.