Studying dynamic characteristics of retinal vessels, as opposed to static measures, may provide additional insight into pathophysiological changes associated with local and systemic abnormalities such as glaucoma and hypertension. Various approaches have been developed to derive static biomarkers from retinal still images, but not many for dynamic analysis of video sequences. This study presents a novel method for the assessment of time-dependent diameter changes in high-speed videos (125 fps) from the rat retina. The proposed method is composed of a vessel segmentation and a diameter measurement module. The specificity and sensitivity of the segmentation method over 25 images were 95.1% and 97.3% respectively. The mean and standard deviation of the diameter measurement errors were -0.147±0.41 pixels over 100 measurements.