Traditional methods of intraoperative human saphenous vein preparation for use as bypass grafts can be deleterious to the conduit. The purpose of this study was to characterize acute graft preparation injury, and to mitigate this harm via an improved preparation technique. Porcine saphenous veins were surgically harvested (unprepared controls, UnP) and prepared using traditional (TraP) and improved preparations (ImP). The TraP used unregulated radial distension, marking with a surgical skin marker and preservation in heparinized normal saline. ImP used pressure-regulated distension, brilliant blue FCF-based pen marking and preservation in heparinized Plasma-Lyte A. Rings from each preparation were suspended in a muscle bath for characterization of physiologic responses to vasoactive agents and viscoelasticity. Cellular viability was assessed using the methyl thiazolyl tetrazolium (MTT) assay and the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay for apoptosis. Contractile responses to potassium chloride (110 mM) and phenylephrine (10 µM), and endothelial-dependent and -independent vasodilatory responses to carbachol (0.5 µM) and sodium nitroprusside (1 µM), respectively, were decreased in TraP tissues compared to both UnP and ImP tissues (p <= 0.05). TraP tissues demonstrated diminished viscoelasticity relative to UnP and ImP tissues (p <= 0.05), and reduced cellular viability relative to UnP control (p <= 0.01) by the MTT assay. On the TUNEL assay, TraP tissues demonstrated a greater degree of apoptosis relative to UnP and ImP tissues (p <= 0.01). In conclusion, an improved preparation technique prevents vascular graft smooth muscle and endothelial injury observed in tissues prepared using a traditional approach.