Background: Injury to the saphenous vein endothelium during harvest impacts patency after coronary artery bypass graft surgery. Many centers are adopting endoscopic saphenous vein harvest (ESVH) instead of using the traditional open saphenous vein harvest (OSVH) technique. Our objective was to compare the effects of ESVH and OSVH on the structural and functional viability of saphenous vein endothelium using multiphoton imaging, immunofluorescence, and biochemical techniques. Methods: Ten patients scheduled for coronary artery bypass graft surgery were prospectively identified. Each underwent ESVH for one portion and OSVH for another portion of the saphenous vein. A 1-cm segment from each portion was immediately transported to the laboratory for processing. The vessel segments were labeled with fluorescent markers to quantify cell viability (esterase activity), calcium mobilization, and generation of nitric oxide. Samples were also labeled with immunofluorescent antibodies to visualize caveolin, endothelial nitric oxide synthase, von Willebrand factor, and cadherin, and extracted to identify these proteins using Western blot techniques. All labeling, imaging, and image analysis was done in a blinded fashion. Results: Esterase activity was significantly higher in the OSVH group (p < 0.0001). Similarly, calcium mobilization and nitric oxide production were significantly greater in the OSVH group (p = 0.0209, p < 0.0001, respectively). Immunofluoresence and Western blot techniques demonstrated an abnormal alteration in distribution of caveolin and endothelial nitric oxide synthase in the ESVH group. Conclusions: Our study indicates that ESVH has a detrimental effect on the saphenous vein endothelium, which may lead to decreased graft patency and worse patient outcomes.