BACKGROUND: Walking tests, like the 2-minute fast walk test, are simple, inexpensive performance-based tests, and therefore seem attractive to estimate the aerobic fitness in people with chronic diseases. OBJECTIVE: To determine the criterion validity of the 2-minute fast walk test for estimating aerobic capacity in patients with Multiple Sclerosis (MS), by comparing it with the peak oxygen uptake (VO2peak in mL/kg/min) as measured by Cardiopulmonary Exercise Testing (CPET) on a cycle ergometer. METHODS: The 2min fast walk test was performed on a marked indoor trajectory, using a static start protocol. Aerobic capacity (VO2peak, in mL/kg/min) was derived from CPET on a cycle ergometer. Criterion validity was tested by means of Pearson's correlation coefficient and should be at least 0.70 for a good criterion validity of the 2min walk test. Linear regression analysis was applied to more precisely estimate VO2peak. RESULTS: In total 141 people with severe MS-related fatigue (mean age 47.0 years (range 23-68 years), 73% women, median disease duration 7.8 years (range 0.3 - 28.7 years)) performed both tests. The distance walked in two minutes ranged from 52.0 to 290.0m (mean 175.1m, sd 44.9m), while the VO2peak varied between 11.31 and 40.28mL/kg/min (mean 22.52mL/kg/min, sd 6.07mL/kg/min). The correlation between the 2min walk test and VO2peak was 0.441 (95% CI: 0.309-0.570). The absolute residual error in estimated VO2peak was 5.47mL/kg/min. CONCLUSIONS: Due to the poor correlation found between the 2min walk test and VO2peak, the 2-min walk test cannot be recommended as a valid alternative for estimating aerobic capacity in persons with MS.