Background: Phase-contrast cine magnetic resonance imaging (PC-MRI) has been used to measure cerebrospinal fluid (CSF) flow dynamics, but the influence of the area of the aqueduct and region of interest (ROI) on quantification of stroke volume (SV) has not been assessed. Purpose: To assess the influence of the area of the ROI in quantifying the aqueductal SV measured with PC-MRI within the cerebral aqueduct. Material and Methods: Nine healthy volunteers (mean age = 29.6 years) were enrolled in the study, and brain MRI examinations were performed on a 3.0-T system. Quantitative analysis of the aqueductal CSF flow was performed using manual ROI placement. ROIs were separately drawn for each of the 12 phases of the cardiac cycle, and changes in aqueduct size during the cardiac cycle were determined. The SV was calculated using 12 different aqueductal ROIs and compared with the SV calculated using a fixed ROI size. Results: There was variation in the size of the aqueduct during the cardiac cycle. In addition, the measured SV increased with a greater area of the ROI. A significant difference in the calculated SVs with the 12 variable ROIs was observed compared with that using a fixed ROI throughout the cardiac cycle. Conclusion: To establish reliable reference values for the SV in future studies, a variable ROI should be considered.