Abstract Objectives: The primary objective was to determine tolerability of ω-3 fatty acids (2150 mg of eicosapentaenoic acid (EPA) and 1050 docosahexaenoic acid (DHA) ethyl esters) vs placebo in women in undergoing a behavioral weight loss intervention (6 months loss and 6 months maintenance). Secondary objectives were to explore potential differences in modulation of blood and benign breast tissue risk biomarkers, satiety and quality of life indices, and weight loss. Results: 46 peri and postmenopausal women were randomized and 42 completed the 6 months of the weight loss intervention and were biomarker evaluable (22 placebo and 20 ω-3 FA). Median baseline BMI in the 42 evaluable women was 31 kg/m2 with a median 6-month relative weight loss of 11% and relative fat mass loss of 20% (DXA). Median 12-month relative mass loss was 10% in the 35 women completing 12 months of the intervention. ω-3 fatty acids increased the ratio of (EPA+DHA): arachidonic acid 2.6-fold (median, range 1.8 - 3.8) vs no change for placebo. There was no difference by randomization group in relative weight or fat mass loss at 6 or 12 months, grade 2 and 3 adverse events, early discontinuation, satiety or other quality of life measures. More serum biomarkers exhibited significant within-group improvement at 6 and 12 months for evaluable women randomized to ω-3 FA than to placebo. At 6 months, significant change (P<0.05) was observed for adiponectin, leptin, adiponectin:leptin ratio, insulin, lipocalin-2, resistin, PAI-1, HGF, CRP, SHBG, and bioavailable testosterone in women randomized to ω-3 FA but only for leptin, adiponectin: leptin ratio and SHBG in those randomized to placebo. At 6 months, the 21 women who lost >10% weight (median 15%) showed significant within-group improvement in adiponectin, leptin, adiponectin:leptin ratio, insulin, lipocalin-2, resistin, PAI-1, HGF, CRP, SHBG, bioavailable estradiol and bioavailable testosterone. For women with <10% weight loss (median 6%) there was significant within-group improvement only for leptin, the adiponectin: leptin ratio, and SHBG. Little change was observed for inflammatory cytokines IL-6, TNF-alpha, MCP-1 or FABP4, or FGF-21 with ω-3 FA or >10% weight loss. Given the dramatic effect of weight loss on biomarkers, we examined within-group and between-group change from baseline to 6 and 12 months for the four subgroups (10-11 women in each) defined by ω-3 FA or placebo and < or > 10% weight loss at 6 months. The subgroup of >10% loss + ω-3 FA had the greatest within-group change in the proportion of significantly modulated biomarkers at 6 months. >10% loss + ω-3 FA was the only subgroup with a significant within-group increase in adiponectin at both 6 and 12 months and achievement of a beneficial ratio of adiponectin (ug/ml) to leptin (ng/ml) of > 1.0 in 100% of participants. There was a significant between-group effect for adiponectin for >10% loss + ω-3 FA vs each of the other groups. Biomarkers were assessed in tissue acquired by random periareolar fine needle aspiration (RPFNA). There were no significant differences in change in cytomorphology or Ki-67 between women randomized to ω-3 FA or placebo but there were significant within-group increases in benign breast adiponectin (pg/ug protein) at 12 months (p=0.014) for women randomized to ω-3 FA. Conclusions: EPA + DHA ethyl esters (3150 mg/day), added to a behavioral weight loss program in overweight women at increased risk for breast cancer, is well-tolerated and may further improve risk biomarker modulation. The increase in adiponectin when ω-3 FA is added to weight loss is of particular interest given that adiponectin opposes the oncogenic effect of leptin and is associated with improved insulin sensitivity and reduced mTOR signaling. Further study is warranted with enough subjects to detect between-group differences. Citation Format: Carol J Fabian, Christie A Befort, Debra K Sullivan, Susan E Carlson, Jennifer L Nydegger, Amy L Kreutzjans, Kandy R Powers, Teresa A. Phillips, Trina Metheny, Carola M Zalles, Erin D Giles, Stephen D Hursting, Bruce F Kimler. Randomized trial of 12 months of omega-3 fatty acids vs placebo during a weight loss intervention in post-menopausal women at increased risk for breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD11-02.