Objective Endothelial dysfunction is an early process in the pathogenesis of atherosclerosis. Transgenic mouse models are widely used to study the development of endothelial dysfunction and atherosclerosis. However, a non-invasive in vivo measurement to analyse vascular function in mice is missing. Methods and Results Transluminal optical coherence tomography (OCT) imaging provides high resolution images of the saphenous artery in their natural surroundings of the vascular bed. To determine endothelial vessel function, we set a vessel clamp and analysed the changes in vessel diameter after release of the occlusion. In C57BL/6J mice, a significant increase in flow-mediated dilation was detectable. We did choose LDL receptor knockout mice as an atherosclerosis-prone model to study flow-mediated dilation by OCT. The saphenous artery of these mice showed a significant reduced flow-mediated dilation compared to wild-type mice, verifying an endothelial dysfunction. Furthermore, we confirmed our findings by ex vivo analysis in the Aorta thoracalis by classical Mulvany Myograph analysis. The reproducibility of results in both different assays was tested in a Bland-Altman plot. Conclusion Changes in vessel diameter could be detected by optical coherence tomography. We provide a novel method for the visualization and quantification of endothelial function in a mouse model in vivo. Our new approach allows analysing progression and reversibility of endothelial dysfunction in the same animal for a prolonged period of time in vivo.