The presence of epithelial cells in breast nipple aspirate fluid (NAF), irrespective of abnormality, has been associated with increased risk of breast cancer in previous studies. We sought to investigate whether the presence of epithelial cells in NAF is associated with nutritional parameters among 71 healthy premenopausal women who participated in the Nutrition and Breast Health Study and provided any samples of NAF during the study. Total of 142 samples which were obtained over a 1-year period of intervention with low-fat and/or high vegetable-fruit diets were available for cytological evaluation. The odds ratios (ORs) and 95% confidence intervals (CIs) for the detection of epithelial cells in NAF were estimated by fitting generalized estimating equations models by quartile level of nutritional parameters. The probability of yielding epithelial cell-positive NAF progressively increased with increasing total fat intake (p = 0.001). The OR for the highest quartile level of fat intake, compared with lowest, was 7.22 (95% CI 1.14-45.82). On the other hand, there were a marginally significant inverse association with total fiber intake as well as an weak inverse association with the number of servings of fruit and vegetables. Furthermore, the probability of detecting epithelial cells in NAF decreased with increasing plasma levels of lutein and α-carotene (p-values for linear trend; 0.001 and 0.049, respectively). The ORs for the highest versus lowest quartile levels are 0.17 (95% CI 0.04-0.65) and 0.19 (95% CI 0.04-0.91), respectively. These results are generally in support of roles of nutritional factors in breast cancer and thus further studies are warranted.