To establish an alternative method, sequential and diameter response analysis (SDRA), to determine dynamic retinal vessel responses and their time course in serial stimulation compared with the established method of averaged diameter responses and standard static assessment.SDRA focuses on individual time and diameter responses, taking into account the fluctuation in baseline diameter, providing improved insight into reaction patterns when compared with established methods as delivered by retinal vessel analyzer (RVA) software. SDRA patterns were developed with measurements from 78 healthy nonsmokers and subsequently validated in a group of 21 otherwise healthy smokers. Fundus photography and retinal vessel responses were assessed by RVA, intraocular pressure by contact tonometry, and blood pressure by sphygmomanometry.Compared with the RVA software method, SDRA demonstrated a marked difference in retinal vessel responses to flickering light (P < 0.05). As a validation of that finding, SDRA showed a strong relation between baseline retinal vessel diameter and subsequent dilatory response in both healthy subjects and smokers (P = 0.001). The RVA software was unable to detect this difference or to find a difference in retinal vessel arteriovenous ratio between smokers and nonsmokers (P = 0.243). However, SDRA revealed that smokers' vessels showed both an increased level of arterial baseline diameter fluctuation before flicker stimulation (P = 0.005) and an increased stiffness of retinal arterioles (P = 0.035) compared with those in nonsmokers. These differences were unrelated to intraocular pressure or systemic blood pressure.SDRA shows promise as a tool for the assessment of vessel physiology. Further studies are needed to explore its application in patients with vascular diseases.