Fluid can be aspirated from the nipples of most non-lactating women. This nipple aspirate fluid (NAF) is a potential source for the discovery of new breast cancer biomarkers. NAF has two distinct protein profiles. Type I NAF is similar to the fluid associated with cystic disease of the breast, whereas type II NAF is enriched in milk-associated proteins. The prevalence of these two profiles differs in healthy women and in breast cancer patients. This study investigated the relationship of these two NAF profiles to reproductive history, body composition, diet, and levels of lipids, steroids and thyroid hormones in healthy premenopausal women (age 30-40 years) who had regular menstrual cycles and normal mammograms and were not taking contraceptive medications. On average, women with the type I NAF profile were older, had more years since last childbirth, were less likely to have breastfed their babies and had higher dietary saturated fat intake, body mass index, body fat mass, and levels of plasma low density lipoproteins than women with the type II profile (P <0.05). Using multiple logistic regression, type I NAF was predicted independently (P <0.05) by higher body fat mass [Odds Ratio (OR) = 3.0; 95% Confidence Interval (CI): 1.5-6.1], more years since last childbirth (OR = 2.6; 95% CI: 1.3-5.2) and a higher percentage of calories from saturated fat (OR = 4.1; 95% CI: 1.1-14.6). These results suggest that protein profiles of NAF might be influenced by amounts or types of dietary and body fat, but further study of the relationship of the two profiles to breast cancer risk is needed.