Objective: We tested the effects of weight loss on serum estradiol, estrone, testosterone and sex hormone binding globulin (SHBG) in overweight/obese women 18-months after completing a year-long, 4-arm, randomized-controlled dietary weight loss and/or exercise trial. Methods: From 2005-2008, 439 overweight/obese, postmenopausal women (BMI>25 kg/m 2), 50-75 years, were randomized to a year-long intervention: diet (reduced calorie, 10% weight-loss, N=118), exercise (225 min/week moderate-to-vigorous activity, N=117), combined diet+exercise (N=117), or control (N=87). At 12-months, 399 women provided blood; of these, 156 returned at 30 months and gave a blood sample. Hormones and SHBG were measured by immunoassay. Changes were compared using generalized estimating equations, adjusting for confounders. Results: At 30-months, participants randomized to the diet+exercise intervention had statistically significant increases in SHBG levels vs. controls (P=0.001). There was no statistically significant change in SHBG in the exercise or diet intervention arms. Hormone levels did not vary by intervention arm from baseline to 30-months. Participants who maintained weight loss at 30months had statistically significantly greater decreases in free estradiol and free testosterone (P trend =0.02, P trend =0.04, respectively) and increases in SHBG (P trend <0.0001) vs. those who did not have sustained weight loss. Levels of other analytes did not vary by weight loss at 30 months. Conclusions: Sustained weight loss results in reductions in free estradiol and testosterone and increases in SHBG 18-months post-intervention.