The Six Spot Step Test (SSST) extends traditional walking outcomes in persons with multiple sclerosis (PwMS) by further challenging components of coordination and balance. Nonetheless, the test-retest agreement of the SSST has not been investigated. To determine the within-day, day-to-day, and inter-rater agreement and reliability of the SSST in PwMS. A secondary aim was to investigate the validity of handheld timing. A total of 38 PwMS with an Expanded Disability Status Scale (EDSS) <6.5 completed two SSSTs with a 5-minute break in-between. After 2 days, this procedure was repeated. Bland-Altman analysis was performed to determine the 95% Limits of Agreement (LOA) and Intraclass Correlation Coefficient (ICC) was calculated. In a subgroup of 18 PwMS, the SSSTs were video-recorded and timed by a second investigator. The relative LOA within and between days were ±15% and ±19%, while ICC were 0.987 and 0.983, respectively. A minor learning effect was found over four tests. The handheld timing error was ±0.5 seconds when compared to video-based timing. The SSST has an acceptable within- and between-day agreement and reliability. For interventional purposes, a change of >19% can be regarded as a real change. Valid timing can be performed by a handheld stopwatch.