Salivary Cortisol and Alpha-Amylase in Infants Aged 0–6 Months Across Different Feeding Types: A Cross-Sectional Study

Early infancy represents a critical period for neuroendocrine development, during which nutrition may influence physiological regulatory systems. Salivary cortisol, reflecting hypothalamic–pituitary–adrenal axis activity, and salivary alpha-amylase (sAA), reflecting sympathetic nervous system activity, are noninvasive biomarkers commonly used to assess physiological processes related to stress. This study aimed to examine whether feeding type (exclusive breastfeeding, exclusive formula feeding, or mixed feeding) was associated with salivary cortisol and sAA levels in infants aged 0–6 months and to explore the relationship between these biomarkers. A cross-sectional study was conducted among 111 healthy infants attending primary health care centers. Infants were categorized according to feeding type. Saliva samples were collected in the morning and analyzed for cortisol and sAA using enzyme-linked immunosorbent assays. Group comparisons were performed using nonparametric tests, Pearson correlation analysis, Quade’s nonparametric analysis of covariance, and multiple linear regression. Salivary cortisol levels differed significantly among feeding groups in initial analyses, with higher levels observed among exclusively breastfed infants compared with formula-fed infants ( p < 0.05). However, in the adjusted analysis (Quade’s nonparametric ANCOVA), after adjustment for infant age, gestational week, and birth weight, the association was no longer statistically significant. sAA levels did not differ significantly between feeding groups. A moderate positive correlation was observed between salivary cortisol and sAA levels ( r = 0.360, p < 0.001). These findings suggest that feeding type may be associated with variation in infant cortisol levels during early infancy, while sAA levels appear less sensitive to feeding-related differences. Further longitudinal studies are needed to clarify how early nutrition relates to the development of infant stress-regulation systems.