BACKGROUND: Physical inactivity in combination with poor nutrition promotes obesity and is the second leading cause of death in the US. It is well known that physical activity (PA) mitigates the incidence of obesity related diseases. Yet, most individuals fail to meet daily recommended PA requirements. Available animal literature suggests that one primary factor that regulates PA is sex hormones. PURPOSE: This study examined the effects of a high fat/high sugar (HF/HS) diet on Testosterone (T) and 17β-Estradiol (E2) concentrations in female mice with and without physical activity. METHODS: All procedures were approved by TAMU IACUC. The offspring of SENCAR breeder pairs (Charles River) were weaned and co-caged at three weeks of age and randomly assigned to a HF/HS diet (45% fat/10% fructose drinking water) (n=6) or ad-lib (AL) control diet (10% fat) (n=4). At four weeks of age, mice were provided with running wheel access. Running speed, distance, and duration were recorded daily until termination at 20wks of age. Serum T and E2 concentrations were analyzed via ELISA. A 2-way ANOVA was utilized to assess significant differences. RESULTS: E2 concentrations in the AL fed mice with- and without-PA (0.30±0.03; 0.26±0.03 ng/ml) and in the HF/HS fed mice with- and without-PA (0.28±0.04; 0.27±0.23 ng/ml), were not affected by diet type, but were significantly affected by PA (p=0.03). T concentrations in the AL fed mice with- and without-PA (0.33±0.01; 0.31±0.34 ng/ml) and in the HF/HS fed mice with- and without-PA (0.46±0.22; 0.21±0.14 ng/ml) demonstrated no significant effect of diet or PA. CONCLUSION: In this study neither T nor E2 concentrations were altered by HF/HS diet. However, PA increased the serum concentrations of E2, but not T. Therefore, physical activity can affect estradiol levels independent of diet. ACKNOWLEDGMENTS: This project was funded by the US Army through the Department of Defense projects W81XWH-13-1-0278 (Fuchs-Young) and W81XWH-13-1-0279 (Lightfoot).