Background Many biologically active factors are present in human milk including proteins, lipids, immune factors, and hormones. The milk composition varies over time and shows large inter-individual variability. This study examined variations of human milk immune factors and cortisol concentrations in the first three months post-partum, and their potential associations with maternal psychosocial distress. Methods Seventy-seven healthy mothers with full term pregnancies were enrolled, of which 51 mothers collected morning milk samples at 2, 6 and 12 weeks post-delivery. Maternal psychosocial distress was assessed at 6 weeks post-delivery using questionnaires for stress, anxiety, and depressive symptoms. Immune factors were determined using multiplex immunoassays and included innate immunity factors (IL1β, IL6, IL12, IFNγ, TNFα), acquired immunity factors (IL2, IL4, IL10, IL13, IL17), chemokines (IL8, Groα, MCP1, MIP1β), growth factors (IL5, IL7, GCSF, GMCSF, TGFβ2) and immunoglobulins (IgA, total IgG, IgM). Cortisol was quantified using liquid chromatography-tandem mass spectrometry. A linear mixed effects model was fit to test whether stress, anxiety, and depressive symptoms individually predicted human milk cortisol concentrations after accounting for covariates. Repeated measurement analyses were used to compare women with high (n = 13) versus low psychosocial distress (n = 13) for immune factors and cortisol concentrations. Results Virtually all immune factors and cortisol, with the exception of the granulocyte-macrophage colony-stimulating factor (GMCSF), were detected in the human milk samples. The