Background Endoscopic vein harvest for lower extremity arterial bypass grafting has been questioned due to concern for endothelial damage during procurement. We sought to compare nitric oxide (NO)–mediated endothelial-dependent relaxation (EDR) in vein segments harvested using open surgical techniques (OH) versus endoscopic vein harvest (EH) techniques. Methods Saphenous vein segments were harvested for lower extremity bypass, and a single, minimally handled section of saphenous vein, free of branches, was taken from the end of the graft. Four 4-mm venous ring segments were then cut and mounted on force transducers. Segments were mounted in 37° oxygenated Krebs-Henseleit solution and maximally contracted using KCl. Individual ring segments that did not react to KCl were excluded from the study. Norepinephrine (NE) was used to achieve submaximal contraction. EDR was determined using increasing concentrations of bradykinin (BDK). Endothelial-independent relaxation (EIR) was confirmed using sodium nitroprusside. Two-way analysis of variance (ANOVA) was used to analyze differences between harvest techniques across BDK concentration and a Student's t-test was used to analyze single comparisons. Results Vein segments harvested from patients (n = 13) led to 28 viable rings that exhibited a positive reaction to KCl (11 rings; 5 patients EH vs. 17 rings; 8 patients OH). Both vein groups achieved moderate relaxation to maximal BDK concentration, [10−6 M]; (49.5% EH vs. 40.55% OH, P = 0.270). Analysis by 2-way ANOVA for mean % relaxation for BDK concentration [10−11–10−6 M] showed improved EDR in EH samples compared with OH (P = 0.029). Mean nitrite/nitrate (NO(x)) tissue bath concentration measurements post-BDK were 139.8 nM (EH) vs. 97.2 nM (OH; P = 0.264). Histology and positive factor VIII immunohistochemistry staining provided evidence for the presence of intact endothelium in our sample segments. EIR was preserved and was similar in the two groups. Conclusions Endothelial function is preserved when utilizing endoscopic harvesting techniques. The advantages of minimally invasive vein procurement for lower extremity bypass can be obtained without concern for damaging venous endothelium.