Background: We conducted a pilot study of high dose omega-3 fatty acid (FA) supplementation in pre-menopausal women to determine if risk biomarkers for breast cancer in benign breast tissue sampled by random peri-areolar aspiration (RPFNA) could be favorably modulated and to acquire preliminary data on possible mechanism of action. Methods: 36 pre-menopausal women at increased risk for breast cancer were accrued to a trial of 6-month intervention with 4 g daily of omega-3-acid ethyl esters [1.86 g eicosapentaenoic acid (EPA), 1.5 g docosahexaenoic acid (DHA)]. To date, 31 subjects have completed study with RPFNA performed pre- and post-intervention in the follicular phase of the menstrual cycle and specimens evaluated for cytomorphology and proliferation (Ki-67). FA composition was determined in plasma, red blood cells, and RPFNA specimens. Additional specimens were frozen for assessment of hormones, a panel of 11 adipokines and cytokines, and gene expression. Results: The ratio of (EPA+DHA):Arachidonic Acid (AA) levels increased significantly in plasma and erythrocytes by a median of three-fold. There was a significant decrease in blood EPA+DHA between discontinuation at 6 months and 2 weeks later when RPFNA was performed. Despite that, there was favorable modulation for cytologic evidence of atypia (81% at baseline to 42% at off-study; p=0.003), Masood score (medians of 15 to 14; p=0.001), number of epithelial cells recovered (p=0.004) and Ki-67 expression (p=0.025 for 30 women with any Ki-67 at baseline, medians of 1.9% to 0.9%). Serum assays have been completed for 24 subjects. No statistically significant changes were observed for estradiol, testosterone, progesterone, SHBG, IGF-1, IGFBP-3. There was a trend (p=0.056) towards a decrease in resistin. To date, only two subjects have discontinued the study early; grade 2 or greater gastrointestinal side effects have been reported by only two subjects. Conclusions: Favorable modulation of tissue risk biomarkers, cytologic atypia and proliferation along with good tolerability suggests that high dose omega-3 FA esters should be tested further in a placebo controlled trial. Clinical trial information: NCT01252277.