© 2023 Elsevier B.V.Background: Functional exercise capacity evaluation is crucial to monitor treatment effects and disease progression in people with multiple sclerosis (pwMS). Compared to other tests, the incremental shuttle walking test (ISWT), which more accurately reflects cardiovascular responses, may be more useful for assessing exercise capacity. The aim of the study is to investigate the reliability and validity of the ISWT. Methods: Thirty-six pwMS with an Expanded Disability Status Scale (EDSS) score<4.5 between the ages of 25 and 65 were included in the study. The subjects underwent practice (ISWT-p) before undergoing the test-retest protocol in order to rule out the ISWT learning effect (ISWT-1 – ISWT-2). ISWT-1 and ISWT-2 were administered with a 3–7 day interval for test-retest reliability. Six-minutes walking test (6MWT) were applied for concurrent validity. The EDSS, pulmonary function tests, Fatigue Impact Scale (FIS), respiratory muscle strength [maximum inspiratory and expiratory pressure (MIP-MEP)] measurements were made for convergent validity. Results: ISWT was found to have excellent test-retest reliability with an ICC value of 0.97. The area under the curve value was 0.904 indicating that ISWT has a good performance for predicting disease severity. The moderate correlation between ISWT and 6MWT (rho: 0.68, p<0,001) proved concurrent validity. It was also moderately correlated with EDSS, MEP (rho: -0.58 and 0.47 respectively), weakly correlated with MIP and FIS (rho:0.37 and -0.36, respectively) while not correlated with pulmonary function tests. Conclusion: The ISWT had excellent test-retest reliability, acceptable criterion and construct validity in ambulatory MS patients.