Abstract Investigating atherosclerosis and endothelial dysfunction has mainly become established in genetically modified ApoE^−/− or LDL-R^−/− mice transgenic models. A new AAV-PCSK9DY^DY mouse model with no genetic modification has now been reported as an alternative atherosclerosis model. Here, we aimed to employ this AAV-PCSK9^DY mouse model to quantify the mechanical stiffness of the endothelial surface, an accepted hallmark for endothelial dysfunction and forerunner for atherosclerosis. Ten-week-old male C57BL/6 N mice were injected with AAV-PCSK9^DY (0.5, 1 or 5 × 10¹¹ VG) or saline as controls and fed with Western diet (1.25% cholesterol) for 3 months. Total cholesterol (TC) and triglycerides (TG) were measured after 6 and 12 weeks. Aortic sections were used for atomic force microscopy (AFM) measurements or histological analysis using Oil-Red-O staining. Mechanical properties of in situ endothelial cells derived from ex vivo aorta preparations were quantified using AFM-based nanoindentation. Compared to controls, an increase in plasma TC and TG and extent of atherosclerosis was demonstrated in all groups of mice in a viral load-dependent manner. Cortical stiffness of controls was 1.305 pN/nm and increased (10%) in response to viral load (≥ 0.5 × 10¹¹ VG) and positively correlated with the aortic plaque content and plasma TC and TG. For the first time, we show changes in the mechanical properties of the endothelial surface and thus the development of endothelial dysfunction in the AAV-PCSK9^DY mouse model. Our results demonstrate that this model is highly suitable and represents a good alternative to the commonly used transgenic mouse models for studying atherosclerosis and other vascular pathologies.