Background and aims We tried to circumvent the limitations of standard organ chamber experiments using in vivo optical coherence tomography (OCT) to analyze the vascular function of small arteries in different mouse strains. Methods OCT images were acquired with a two-axis galvanometer scanner head. Time series (3 frames per second, 300 × 512 pixel per frame) of cross-sectional images were analyzed with image processing software measuring the time course of vessel lumen dynamics. Vascular function of murine saphenous artery of male C57BL/6 (wild-type) and hypercholesterolemic LDLR knockout (LDLR^−/−) mice was analyzed at 6 weeks and after 14 weeks feeding a control or high-fat diet containing 21.2% butter fat and 2.1 mg/kg cholesterol. Vasoconstriction and vasodilation was analyzed by OCT in response to 80 mM K⁺ and 1 mM SNP. Results The OCT technique allowed determination of inner diameter, flow resistance, maximal velocity of diameter change and time to half-maximal diameter change in murine saphenous arteries of wild-type and LDLR^−/− mice. LDLR^−/− had impaired vasodilation and changes in vasodynamics after 14 weeks on control or high-fat diet, compared to wild-type mice. The diameter of the saphenous artery of LDLR^−/− mice was reduced after vasoconstriction (38 ± 7 μm vs 12 ± 6 μm) and vasodilation (245 ± 8 μm vs 220 ± 10 μm) (P < 0.05 vs C57BL/6). Conclusion OCT was used as an innovative method to image vascular function of small arteries of wild-type and hypercholesterolemic LDLR^−/− mice after high-fat diet. The method offers the ability to display differences in the vasodynamics at early stages of endothelial dysfunction in vivo.