Abstract
Breastfeeding provides essential bioactive factors such as cortisol and melatonin, critical hormones for establishing infant circadian rhythms, sleep-wake cycles, emotional regulation, and resilience. Cortisol supports immune function and brain development, while melatonin regulates sleep-wake cycles and influences metabolism. The establishment of circadian rhythms for these hormones begins around 9-12 weeks of life, progressing significantly within the first 6 months. Cortisol and melatonin in breast milk play a crucial role in these processes, yet it remains unclear whether their concentrations and timing in milk reflect maternal salivary levels. This study examined the relationship between maternal salivary and milk concentrations of cortisol and melatonin and infant concentrations. Lactating women aged 18+ with infants under 4 months old (n = 15) provided up to 10 maternal saliva, infant saliva, and breast milk samples over 24 hours. Cortisol in saliva and milk was quantified via enzyme-linked immunosorbent assay (ELISA), while melatonin was analyzed using liquid chromatography–mass spectrometry (LC-MS). Infant salivary cortisol mirrored milk concentrations, displaying a two-peak diurnal pattern. Maternal salivary cortisol was significantly lower than milk cortisol (p < 0.01). As expected, melatonin was absent in daytime milk, reinforcing its circadian role. These findings highlight breast milk as a dynamic exogenous cue critical for developing infant circadian rhythms.