Purpose. Detection and precise quantification of changes in retinal vessel diameter by image analysis techniques is important in a number of fields of research. The retinal vessels have been shown to exhibit pulse related changes in diameter. These may need to be taken into account when studying diameter changes due to other causes. This study examined the effect of using multiple fundus photographs with and without electro-cardiographic synchronisation on the size and statistical significance of changes in mean retinal vessel diameter.Methods. Twelve fundus photographs spaced throughout the cardiac cycle by electrocardiographic synchronisation were taken in 10 normal volunteers: (a) at rest, (b) during isometirc exercise, and (c) during oxygen inhalation. Vessel diameters were measured using a computer assited image analysis system. Subsequently smaller sample sizes, with and without electrocardiograph synchronisation were modelled from the available data.Results. With a group of ten subjects six or more electrocardiograph synchronised photographs enabled reliable detection of small diameter changes (1.4%) induced by isometric exercises while other methods either failed to detect change or were unreliable at doing so. With six subjects twelve synchronised photographs were required to reliably detect a change of the same magnitude. Larger diameter changes (5.4%) were detected by any method including a single unsynchronised photograph.Conclusions. Multiple frame electrocardiograph synchronized fundus photography permits more accurate detection of small changes in retinal vessel diameter.