Abstract Body: Nitrate supplements are commonly combined with aerobic exercise to potentially improve performance and cardiac function. Whether this combination benefits cardiac function at the organ and cellular levels is unclear. We will determine if nitrates affect fitness or cardiac function in both sexes. C57Bl/6 mice (9-mos) were given 1mM sodium nitrate (drinking water) and/or access to a running wheel for 3 mos. Cardiac function (echocardiography), blood pressure (tail cuff) and fitness (treadmill) were measured. Calcium transients (Fura-2) and contractions (2Hz) were measured in ventricular myocytes and calcium handling proteins were measured by qPCR. In both sexes, nitrates did not change blood pressure or voluntary wheel running although females ran more than males (3.7±0.3km vs 2.2±0.3km, p=0.002). In exercised female mice, nitrates increased fitness from baseline while control did not (top speed at exhaustion: 24.2±1.4 to 28.0±1.5 cm/s, p=0.07 vs 23.3±1.0 to 30.7±2.0 cm/s, p=0.002) but nitrate supplemented males showed no fitness benefit. However, while exercise improved cardiac function and contractility compared to sedentary mice, nitrates plus exercise impaired cardiac function in a sex-specific manner. Nitrate-treated males who exercised had lower E/A ratios (1.8±0.3 vs 1.3±0.1, p=0.01) and worse diastolic function than control exercised males. By contrast, nitrate-treated female mice who exercised had smaller global longitudinal strain (-19.5±1.5 vs -11.8±3.0%, p=0.01) than controls, indicative of worse systolic function. Corresponding changes were seen at the cellular level. Exercised males given nitrates had slower a calcium transient decay than controls (44.8±4 vs 73.8±6 ms, p=0.01), while females given nitrates had slower velocity to peak calcium (0.031±0.004 vs 0.018±0.003 µm/ms, p=0.005) and smaller cellular contractions (2.9±0.5 µm vs 1.4±0.5 µm: p=0.03). Cardiac calcium handling proteins were also modified in females. Exercise plus nitrates caused a decrease in SERCA2a and ryanodine receptor 2 expression (mRNA) compared to the control exercise group. Studies in males are ongoing. Exercise plus nitrates increased fitness levels in only females and reduced cardiac function in a sex specific way when compared to exercise alone. This suggests a potential balance between benefits to fitness and potential cardiac complications that could affect athletes taking nitrate supplementation.