Consumption of high amounts of saturated fat is a well established risk factor of patients with cardiovascular diseases. An important step in the pathogenesis of arteriosclerosis is the development of endothelial dysfunction. However, induction of endothelial dysfunction by high-fat diet is difficult in mouse models and controversial in the literature. In this study, we show a reproducible development of endothelial dysfunction using a very high-fat diet. C57BL/6J mice were fed a diet containing 60% kcal from fat for 20 weeks. Body weight and the amount of epididymal, retroperitoneal, mesenteric and perivascular fat were significantly increased after high-fat diet. Diet-induced obesity elevated blood glucose, triglyceride, total cholesterol, and low-density lipoprotein plasma levels. Interestingly, cumulative food intake was lower in mice on high-fat diet compared to control. This study is the first showing an analysis of endothelial function by self-developed optical coherence tomography devices using flow-mediated vasodilation in the murine saphenous artery in vivo. In addition, we performed ex vivo analysis of endothelial function in rings of thoracic aortas from these animals using a Mulvany myograph. Both methods showed a significantly impaired endothelium-dependent relaxation. As control, endothelial dysfunction was shown by the same approach in atherosclerotic Ldlr^-/- mice. Interestingly, application of COX-1/2 inhibitor diclofenac inhibited this effect. Next, we studied the role of H₂O₂ in the murine thoracic aorta. Catalase increased EC₅₀ values in endothelial function of mice on standard chow. Moreover, a reduced H₂O₂ release was measured in the thoracic aorta after high-fat diet compared to mice on standard chow. This might contribute to the reduced endothelium-dependent vasodilation after high-fat diet. In conclusion, we were able to induce endothelial dysfunction in a reproducible manner in murine models using a very high-fat diet. This is the first study showing endothelial dysfunction by self-developed optical coherence tomography devices using flow-mediated vasodilation in mice models in vivo. A reduced release of H₂O₂ might contribute to the impaired endothelium-dependent vasodilation after high-fat diet.