peripheral arterial disease (pAD) develops due to the narrowing or blockage of arteries supplying blood to the lower limbs. Surgical and endovascular interventions are the main treatments for advanced pAD but alternative and adjunctive medical therapies are needed. currently the main preclinical experimental model employed in pAD research is based on induction of acute hind limb ischemia (HLI) by a 1-stage procedure. Since there are concerns regarding the ability to translate findings from this animal model to patients, we aimed to develop a novel clinically relevant animal model of pAD. HLi was induced in male Apolipoprotein e (ApoE −/− ) deficient mice by a 2-stage procedure of initial gradual femoral artery occlusion by ameroid constrictors for 14 days and subsequent excision of the femoral artery. This 2-stage HLI model was compared to the classical 1-stage HLI model and sham controls. ischemia severity was assessed using Laser Doppler perfusion imaging (LDpi). Ambulatory ability was assessed using an open field test, a treadmill test and using established scoring scales. Molecular markers of angiogenesis and shear stress were assessed within gastrocnemius muscle tissue samples using quantitative polymerase chain reaction. HLI was more severe in mice receiving the 2-stage compared to the 1-stage ischemia induction procedure as assessed by LDPI (p = 0.014), and reflected in a higher ischemic score (p = 0.004) and lower average distance travelled on a treadmill test (p = 0.045). Mice undergoing the 2-stage HLI also had lower expression of angiogenesis markers (vascular endothelial growth factor, p = 0.004; vascular endothelial growth factor-receptor 2, p = 0.008) and shear stress response mechano-transducer transient receptor potential vanilloid 4 (p = 0.041) within gastrocnemius muscle samples, compared to animals having the 1-stage HLI procedure. Mice subjected to the 2-stage HLI receiving an exercise program showed significantly greater improvement in their ambulatory ability on a treadmill test than a sedentary control group. this study describes a novel model of HLi which leads to more severe and sustained ischemia than the conventionally used model. Exercise therapy, which has established efficacy in PAD patients, was also effective in this new model. this new model maybe useful in the evaluation of potential novel pAD therapies.Peripheral arterial disease (PAD) leads to impaired lower limb blood supply usually as a result of atherosclerosis and associated thrombosis 1 . In 2010 an estimated 200 million people worldwide were living with PAD, an increase of ~30% since 2000 which has been referred to as a PAD pandemic 2,3 . The burden of PAD is increasing worldwide and particularly within low and middle-income countries 4 . PAD is associated with significant morbidity including intense leg pain during walking (intermittent claudication, IC), impaired walking ability, poor health-related quality of life and risk of serious complications such as major leg amputation and death 1 . Medical management of PAD is ...