INTRODUCTION: Arterial stiffening is a hallmark of vascular ageing, and unravelling its underlying mechanisms has become a central theme in the field of cardiovascular disease. While various techniques and experimental setups are accessible for investigating biomechanics of blood vessels both in vivo and ex vivo, comparing findings across diverse methodologies is challenging. METHODS: Arterial stiffness in the aorta of adult (5 months) and aged (24 months) wild-type C57Bl/6J mice was measured in vivo, after which ex vivo biomechanical evaluation was performed using the Rodent Oscillatory Tension Setup to study Arterial Compliance (ROTSAC
University of Antwerp, Belgium) and the DynamX setup (Maastricht University, The Netherlands). Stiffness of aortic tissue was measured in both absence and presence of activated smooth muscle cells (i.e., contraction). Measurements in both setups were conducted in parallel with matched protocols and identical buffers and chemicals. RESULTS: Overall, both methods revealed age-related increased aortic stiffness, although parameters of aortic mechanics showed different numerical values, suggesting that results are not directly interchangeable between methods. Surprisingly, smooth muscle cell contraction had opposing effects between the setups. Indeed, smooth muscle cell contraction increased arterial stiffness in the ROTSAC but decreased stiffness in the DynamX. These opposing effects could be attributed to how the two setups differentially load the collagen fibres in the arterial wall, ex vivo. CONCLUSION: Overall, this study provided critical insights into how different experimental setups can influence the interpretation of aortic biomechanics, emphasizing the need for careful consideration and contextualization of results based on the methodology used.