Although continuous perfusion of donor hearts for preservation during transportation has been widely applied, intermittent perfusion has been suggested as an alternative. The aim of this study was to identify the optimal intermittent perfusion protocol by investigating the effects of perfusion volume on endothelial and inflammatory marker expression by the coronary artery. Donor porcine hearts were perfused with various volumes of Celsior solution supplemented with diazoxide (50, 100, 150, 200, and 250 ml) every 2 h for 30 min each for a 10 h period. The effects on cardiomyocytes and vascular endothelial cell morphology and marker expression were compared to the immersion control group. Whereas an incomplete endothelial cell layer with disorganized connective tissue was observed in the control and 50, 100, and 150 ml intermittent perfusion groups, transmission electron microscopic analysis revealed a complete endothelial cell layer in the intima with an organized subendothelium. A perfusion volume-dependent increase in eNOS expression that coincided with a decrease in ET-1, ICAM-1, vWF, and P-selectin expression was detected (all p < 0.01). Intermittent perfusion with 200 ml of Celsior solution every 2 h conferred protective effects simultaneously to the coronary arteries and myocardium on the porcine donor heart over a clinically relevant preservation period.