Abstract Body (Do not enter title and authors here): BACKGROUND: Cardiovascular disease (CVD) is the leading cause of death in men with prostate cancer (PC), and vascular function and arterial stiffness can predict CVD. High circulating free testosterone has been associated with increased arterial stiffness in PC, and higher circulating free testosterone early in prostate cancer diagnosis is associated with more aggressive disease. However, the role of circulating free testosterone and its influence on microvascular function in men with PC has yet to be elucidated. Thus, the present study sought to investigate the relationship between free testosterone and a comprehensive assessment of vascular health over time in men with PC. METHODS: Thirty-two men (Age: 67±1 years; BMI: 28.2±1.1 kg/m²) with PC participated in this study. Fasting blood samples were collected to assess both total and free testosterone. The flow-mediated dilation (FMD) test was used to assess conduit vessel function and microvascular function was assessed using local thermal heating (LTH). Arterial stiffness was assessed using augmentation index (AIx75). Blood samples and assessments of vascular health were conducted at baseline and every 3 months thereafter. Data are reported as mean±SEM. RESULTS: Free testosterone decreased (p=0.079) from baseline (5.0±0.5 pg/mL) to 3 months (4.6±1.1 pg/mL) and remained lower at 6 months (4.4±0.7 pg/mL), and 9 months (4.7±0.8 pg/mL). In addition, total testosterone was similar (p>0.05) from baseline (318±38 ng/dL) throughout the study. Free testosterone at baseline was inversely correlated with the change in LTH at 3 months (r=-0.601; p=0.030), 6 months (r=-0.638; p=0.035), and 9 months (r=-0.671; p=0.048). There was a positive correlation between free testosterone at baseline and the change in AIx75 from baseline to 3 months (r=0.668; p=0.013). No relationships between free testosterone and FMD over time were identified, nor were any relationships observed between total testosterone and indices of vascular health. DISCUSSION: Findings from this study demonstrate that a higher circulating free testosterone at baseline is associated with increased microvascular dysfunction and arterial stiffness over time. Future studies are needed to elucidate the precise mechanistic role of free testosterone on microvascular function in prostate cancer and whether therapeutic approaches can be used to mitigate the negative cardiovascular effects associated with lower concentrations of androgens.