INTRODUCTION: Arterial stiffness (AS) is a prevalent condition associated with increased risk of cardiovascular (CV) complications. Research has shown structural changes associated with AS, for which the precise mechanisms are unknown. Mechanical properties testing (MPT) has the potential to provide insight into the pathophysiology of AS, but studies to date have not represented physiological conditions well, limiting their contribution. PURPOSE: To develop a MPT method that overcomes these limitations. METHODS: Four groups of Sprague Dawley rats were studied: Control (C), acute diabetes (DA), chronic diabetes (DC) and insulin treated chronic diabetes (DI). Following sacrifice, each aorta was excised and cut into 4 rings. Rings were tested using cyclic and failure testing protocols. RESULTS: DI low (p<0.05) and high pressure (p<0.05) groups had significantly greater wall thickness than all other groups. DI low pressure first peak strain was lower than DA and DC low pressure groups (p<0.05) but was not different between high pressure groups. Modulus was not different between low pressure groups, but DI high pressure group modulus was significantly lower than all other groups (p<0.05). During cyclic testing, stress difference was significantly lower in DI low pressure than DC and C low pressure groups (p<0.05) and significantly lower in DI high pressure compared to all other high pressure groups (p<0.05). Strain difference was not different between groups. DISCUSSION: Our findings suggest that DI group aortas were weaker compared to the other groups. Previous research suggests that insulin therapy can induce structural alterations in the vascular wall which poorly affect cardiovascular health. The new method of MPT used in this study was successful in measuring aortic stiffness differences between groups and may be a useful tool for future studies investigating responsible mechanisms of AS development in other disease states.