Background: Several small cross-sectional studies have investigated cerebrospinal fluid (CSF) flow dynamics in multiple sclerosis (MS) patients and have reported mixed results. Currently, there are no longitudinal studies that investigate CSF dynamics in MS patients. Objective: To determine longitudinal changes in CSF dynamics measured at the level of aqueduct of Sylvius (AoS) in MS patients and matched healthy controls (HCs). Materials and methods: Forty (40) MS patients and 20 HCs underwent 3T MRI cine phase contrast imaging with velocity-encoded pulse-gated sequence at baseline and 5-year follow-up. For atrophy determination, MS patients underwent additional high-resolution 3D T1-weighted imaging. Measures of AoS cross-sectional area (CSA), average systolic and diastolic velocity peaks, maximal systolic and diastolic velocity peaks and average CSF flow rates were determined. Brain atrophy and ventricular CSF (vCSF) expansion rates were determined. Cross-sectional and longitudinal changes were derived by analysis of covariance (ANCOVA) and paired repeated tests. Confirmatory general linear models were also performed. False discovery rate (FDR)-corrected p-values lower than 0.05 were considered significant. Results: The MS population demonstrated significant increase in maximal diastolic peak (from 7.23 to 7.86 cm/s, non-adjusted p = 0.037), diastolic peak flow rate (7.76 ml/min to 9.33 ml/min, non-adjusted p = 0.023) and AoS CSA (from 3.12 to 3.69 mm 2 , adjusted p = 0.001). The only differentiator between MS patients and HCs was the greater AoS CSA (3.58 mm 2 vs. 2.57 mm 2 , age-and sex-adjusted ANCOVA, p = 0.045). The AoS CSA change was associated with vCSF expansion rate (age-and sex-adjusted Spearman's correlation r = 0.496, p = 0.019) and not with baseline nor change in maximal velocity. The expansion rate of the vCSF space explained an additional 23.8% of variance in change of AoS CSA variance when compared to age and sex alone (R 2 = 0.273, t = 2.557, standardized β = 0.51, and p = 0.019). Conclusion: MS patients present with significant longitudinal AoS enlargement, potentially due to regional atrophy changes and ex-vacuo expansion of the aqueduct.