Abstract Body: INTRODUCTION: Peripheral artery disease (PAD) is a major form of atherosclerotic cardiovascular disease. Women with PAD typically present clinically at older ages after the onset of menopause; however few preclinical studies have attempted to model this condition. Herein, we tested the effects of surgical ovariectomy (OVX) and 4-vinylcyclohexene diepoxide (VCD), a chemical that induces gradual ovarian failure, on the development of atherosclerosis and the response to hindlimb ischemia (HLI). We hypothesized that 4-VCD and OVX would enhance the development of atherosclerosis and worsen ischemic limb outcomes compared to normally cycling female mice. METHODS: Twelve-week-old female C57BL/6J mice were given a single tail vein injection of AAV8 encoding a gain-of-function mutation of proprotein convertase subtilisin/kexin type9 (AAV8-PCSK9) and were fed an atherogenic diet for 24 weeks. Based on baseline body weight, mice were randomized to control (CON: n=12), 4-VCD treatment (20-day consecutive injections at 160 mg/kg; n=11), or OVX (performed at Week15; n=12). Blood samples were collected every four weeks to measure total, LDL, and HDL, cholesterol levels. Femoral artery ligation (FAL) was performed at Week 20, and assessments of hindlimb function and pathology were performed 28 days post-FAL. RESULTS: Compared to baseline, total cholesterol increased 4.9-, 4.7-, and 5.1-fold in CON, 4-VCD, and OVX mice. Similar trends were observed for LDL (4.1-, 4.0-, and 4.5-fold) and HDL cholesterol (2.3-, 2.2-, and 2.3-fold). Treatment group had no effect on total (P=0.762), LDL, (P=0.305), and HDL (P=0.375) cholesterol levels. The atherosclerotic lesion area in the thoracic aorta was greater in 4-VCD (P=0.006; 17.9±1.7%) and OVX (P=0.035; 16.5±1.3%) than CON (10.8±1.5%) but was similar between 4-VCD and OVX (P=0.790). Following FAL, perfusion recovery was not different between groups (P=0.321). Plantarflexor muscle weight was similar across groups in both ischemic (P=0.175) and non-ischemic (P=0.236) limbs, and HLI did not cause muscle atrophy (P=0.083). Muscle strength of ischemic limb normalized to non-ischemic limb was 16.4% lower in 4-VCD than CON (P=0.034), with no difference between 4-VCD and OVX (P=0.236). CONCLUSION: While 4-VCD- or OVX- induced menopause had minimal impacts on blood lipid profiles and limb pathophysiology following FAL, both gradual ovarian failure and surgical OVX exacerbated atherosclerosis in female mice.