Pulsatile changes in retinal vascular geometry over the cardiac cycle have clinical implication for diagnosis of ocular and systemic vascular diseases. In this study, we report a Vesselness Mapping of Retinal Image Sequence (VMRS) methodology to visualize the vessel pulsation and quantify the pulsatile motions in the cardiac cycle. Retinal images were recorded in an image sequence corresponding to 8 segments of the cardiac cycle using a nonmydriatic fundus camera (Canon CR45, Canon Inc., Japan) modified with ECG-synchronization. Individual cross-sectional vessel diameters were measured separately and the significance of the variations was tested statistically by repeated measures analysis of variance (ANOVA). The graders observed an improved quality of vessel pulsation on a wide region around the optic disk using the VMRS. Individual cross- sectional vessel diameter measurement after visualization of pulsatile motions resulted in the detection of more significant diameter change for both arterioles (3.3 μm, P = 0.001) and venules (6.6 μm, P < 0.001) compared to individual measurement without visualization of the pulsatile motions (all P values > 0.05), showing an increase of 2.1 μm and 4.7 μm for arterioles and venules, respectively.