Abstract Body (Do not enter title and authors here): We have previously shown that chronic exposure to 17β-estradiol promotes microvascular endothelial dysfunction in isolated human arterioles from healthy adult individuals assigned male at birth (IAMB). Microvascular dysfunction is a strong predictor of future cardiac events and women of transgender experience have a higher risk of cardiovascular death, however the effects of continuous gender affirming hormone therapy on in vivo microvascular function remain unknown. We hypothesized that in vivo endothelial-derived, nitric oxide (NO)-mediated vasodilation is impaired within the peripheral microvasculature in IAMB who have been exposed to continuous gender affirming hormone therapy compared to age-matched controls. Inclusion criteria for this prospective, cross-sectional study included healthy IAMB, age 18-60 years, no past medical history of cardiovascular disease, and a minimum 1-year exposure to gender affirming hormone therapy (estrogen; E2). Peripheral microvascular function was assessed using Laser Doppler Flowmetry with microdialysis (LDF-MD). Two microdialysis fibers were placed in the ventral forearm of eleven subjects (3 IAMB, 8 IAMB+E2) with sites randomized to serve as 1) control (lactated Ringer’s) and 2) infusion with the nitric oxide synthase inhibitor L-NAME. After baseline measurement, vehicle and L-NAME were administered followed by infusion of two doses of acetylcholine (Ach; 1mM, 10mM). Following a rest period, a local heating protocol was then implemented to assess NO-mediated dilation and maximal vasodilation (39°C and 42°C + sodium nitroprusside, respectively). Compared to controls, a reduced trend in dilation to Ach was observed in IAMB+E2 (1mM Ach max dilation (%): 57.2±13.0 vs 80.2±8.7, p=0.33; 10mM Ach max dilation (%): 70.4±7.0 vs 93.7±3.1, p=0.08, IAMB+E2 and IAMB, respectively). A reduced trend in dilation was also observed in IAMB+E2 compared to controls in response to local heating (32.9±8.9 % vs 60.7±6.2 %, respectively, p=0.10). The degree to which dilation was inhibited in the presence of L-NAME was higher with the local heating protocol for the control group (46.6%) vs the estrogen group (22.2%). Together, this pilot study suggests that in vivo microvascular function may be impaired by chronic estrogen therapy in women of trans experience.