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Although the major functions of the lymphatic system are fairly well defined, its vasculature has yet to be well characterized in comparison to its blood vasculature counterpart. Recent advances in optical imaging techniques have allowed for more detailed and quantitative evaluations of lymph flow dynamics and mechanism. A rat tail is often used for investigations of lymph flow because of the simple geometry, superficial nature, and disease progression models of its collecting lymphatic vessels. In this study, a pressure cuff system was fabricated and coupled with an existing functional near infrared (NIR) imaging system to measure the overall pumping pressure of the lymphatic vessels of a rat tail. In addition to adapting the system for use on rodents, previous systems used for measuring lymphatic pumping pressure in humans were improved upon in several ways. The system defined here utilizes closed-loop feedback control of pressure application at smaller, more precise intervals. Using this device, a significant difference in lymphatic vessel pumping pressure was detected between a control case and a treatment case in which a vasoactive substance with a nitric oxide donor (GTNO ointment) was applied to the tail. Although it is known that nitric oxide plays a crucial physiologic role in propagation of flow through lymphatic vessels, this study has quantified its significant pharmacological reduction of pumping pressure for the first time.