Retinal vessel diameter, which is an important parameter in blood flow measurement, is affected by pulsation during the cardiac cycle and by vasomotion. This project studied these changes by analysing three monochromatic fundus photographs taken in eight arbitrary parts of the cardiac cycle of 10 healthy subjects. It was found that the venous diameter decreased in early systole, increasing thereafter to a maximum level in early diastole and then declined towards end diastole. The maximum change of 4.82% (between early systole and early diastole) (p = 0.03) represents a 9.83% change in volumetric blood flow. The arterial diameter peaked in mid-late systole, increasing by 3.46% (p = 0.01); this represents a blood flow increase of 7.04%. Vasomotion led to changes of 3.71% and 2.61% in arteries and veins respectively. It is concluded that for accurate measurement of retinal blood flow, fundus photographs should be taken synchronised with the electrocardiogram.