Accurate sizing of vessel diameter is important for understanding the physiology of blood vessels as well as treatment of coronary and peripheral artery disease. The objective of this study is to validate a novel catheter-based system (LumenRECON, LR) for real-time reconstruction of lumen cross-sectional area (CSA) along the length of a vessel segment. A total of twenty two swine (20 Yorkshire and 2 atherosclerotic Ossabaws) were used to evaluate the accuracy, reproducibility and safety of the system in comparison with intravascular ultrasound (IVUS). The CSA of RCA, LAD and LCx arteries were determined by IVUS and LR over a 3-4 cm segment in twelve Yorkshire, two atherosclerotic Ossabaw swine and two postmortem atherosclerotic human hearts. In eight chronic animals, the effect of LR catheter on the vessel wall was evaluated at 1 day and at 2 weeks (four animals each) after intervention. A Bland-Altman plot of the LR and IVUS show a mean difference between the two measurements of 0.055 mm in diameter which is not statistically significant from zero indicating lack of bias in the comparison of LR to IVUS. The root mean square error of the two measurements was 10.2% of the mean IVUS diameter. The repeatability of the LR system was assessed using duplicate measurements. The mean of the difference between the two measurements was nearly zero and the repeatability coefficient was within 4.5% of the mean of the two measurements. No injury or intimal hyperplasia was found acutely or chronically after the use of LR. This study establishes the accuracy, reproducibility and safety of a non-imaging 2.7Fr sizing catheter in lumen sizing of coronary arteries. The system provides a continuous quantitative axial profile of the mean vessel lumen in real-time and may have significant utility in vascular research and clinically in the catheterization laboratory.