Background: MicroRNAs (miRNAs) target 60% of human messenger RNAs and can be detected in tissues and biofluids without loss of stability during sample processing, making them highly appraised upcoming biomarkers for evaluation of disease. However, reporting of the abundantly expressed miRNAs in healthy samples is often surpassed. Here, we characterized for the first time the physiological miRNA landscape in a biofluid of the healthy breast: nipple aspirate fluid (NAF), and compared NAF miRNA expression patterns with publically available miRNA expression profiles of healthy breast tissue, breast milk, plasma and serum. Methods: MiRNA RT-qPCR profiling of NAF (n = 41) and serum (n = 23) samples from two healthy female cohorts was performed using the TaqMan OpenArray Human Advanced MicroRNA 754-Panel. MiRNA quantification data based on non-targeted or multi-targeted profiling techniques for breast tissue, breast milk, plasma and serum were retrieved from the literature by means of a systematic search. MiRNAs from each individual study were orderly ranked between 1 and 50, combined into an overall ranking per sample type and compared. Results: NAF expressed 11 unique miRNAs and shared 21/50 miRNAs with breast tissue. Seven miRNAs were shared between the five sample types. Overlap between sample types varied between 42% and 62%. Highly ranked NAF miRNAs have established roles in breast carcinogenesis. Conclusion: This is the first study to characterize and compare the unique physiological NAF-derived miRNA landscape with the physiological expression pattern in breast tissue, breast milk, plasma and serum. Breast-specific sources did not mutually overlap more than with systemic sources. Given their established role in carcinogenesis, NAF miRNA assessment could be a valuable tool in breast tumor diagnostics.