Background: Walking disorders represent the most disabling condition in persons with Multiple Sclerosis (PwMS). Several studies showed good reliability of the 6-min walk test (6MWT) (i.e., especially distance traveled), but little is known about the reliability of the Spatio-temporal (ST) variables in the 6MWT. Objective: To evaluate the test-retest reliability of ST variables and perceived exertion during the 6MWT in PwMS and comparable healthy persons. Methods: We explored three 1-min intervals (initial: 0′–1′, middle: 2′30″–3′30″, end: 5′–6′) of the 6MWT. Six ST variables and perceived exertion were measured (respectively, using the GAITRite system and the Borg Scale). These measurements were performed twice, 1 week apart. The test-retest effects were assessed using the intraclass correlation coefficient (ICC) or the weighted kappa. Results: Forty-five PwMS and 24 healthy persons were included. The test-retest reliability of ST variables values was good-to-excellent for PwMS (ICC range: 0.858–0.919) and moderate-to-excellent for healthy persons (ICC range: 0.569–0.946). The test-retest reliability values of perceived exertion were fair for PwMS (weighted kappa range: 0.279–0.376) and substantial for healthy persons (weighted kappa range: 0.734–0.788). Conclusion: The measurement of ST variables during these 6MWT intervals is reliable and applicable in clinical practice and research to adapt rehabilitation care in PwMS.