Studies of lymphatic function often employ collecting lymphatic vessels that exhibit large-amplitude, spontaneous contractions. Data from such preparations have been analyzed using cardiac pump analogies that require accurate determination of vascular dimensions, including external (OD) and internal (ID) diameters. These measurements would be facilitated by an accurate automated measurement system. A computer-based diameter tracking system was developed specifically for lymphatic vessels, with advantages over previous automated systems. The system also permits continuous diameter tracking at two axial locations, enabling the measurement and analysis of contraction wave conduction. The method was validated using spontaneously contracting segments of rat thoracic duct which sometimes exhibited conducted contraction waves. In such preparations, conduction wave velocity was modulated by the axial flow rate and could be easily measured by the tracking system. The method offers improvement and increased convenience over manual diameter measurements in lymphatic vessels, with little or no sacrifice in accuracy. It should be a useful tool for general studies of collecting lymphatic function as well as for the analysis of contraction wave conduction and coordination.