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We aimed to investigate the effect of gender-affirming hormone therapy (GAHT) on cardiovascular disease risk factors focusing on glucose tolerance. This primarily translational study enrolled 16 transgender persons assigned female at birth (AFAB), 22 assigned male at birth (AMAB), and 33 age- and BMI-matched cisgender controls at the Medical University of Vienna from 2013 to 2020. A 3-Tesla MRI scan to measure intramyocardial, pancreatic, hepatic fat content and subcutaneous-to-visceral adipose tissue ratio (SAT/VAT-ratio), an oral glucose tolerance test (oGTT), bloodwork including brain natriuretic peptide (pro-BNP), sex hormones and two glucose-metabolism related biomarkers (adiponectin, betatrophin) were performed. Estrogen intake was associated with higher fasting insulin (p = 0.034) and Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) (p = 0.037), however, lower HbA1c levels (p = 0.031) in AMAB than cisgender males. Adiponectin (p = 0.001) and betatrophin (p = 0.034) levels were higher in AMAB than cisgender males, but similar to cisgender females. Compared to cisgender females, AFAB displayed no differences in glucose metabolism or SAT/VAT-ratio. AFAB had lower pro-BNP levels (p = 0.014), higher liver enzymes (AST: p = 0.011; ALT: p = 0.012) and lower HDL levels (p = 0.017) than cisgender females, but comparable levels to cisgender males. AMAB showed an increased heart rate (p < 0.001) and pro-BNP (p = 0.002) levels, but a more favorable SAT/VAT-ratio (p = 0.013) and lower creatine kinase (CK) (p = 0.001) than cisgender males. There were no relevant differences in organ fat content between transgender persons and their respective cisgender controls. In AMAB, most investigated parameters adapted to levels seen in cisgender females except for parameters related to fasted insulin resistance. AMAB should be monitored with respect to the development of insulin resistance. [ABSTRACT FROM AUTHOR]