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Stress system activity in early life can have long-term effects on neurodevelopment. The main aim of this study was to assess the association of child evening salivary cortisol and alpha-amylase basal levels at 14 months of age with longer-term neuropsychological development at 4 years in a low-risk population-based birth cohort derived from the INMA (Environment and Childhood) project in Spain. We included 186 parent-children pairs with information on both stress system activity and neurodevelopment. Both stress markers at 14 months of age showed an association with neuropsychological development at 4 years. Salivary cortisol showed a sex-specific pattern of association. In girls, cortisol levels at 14 months were negatively associated with cognitive development [long-term declarative memory (β = − 17.8, p = 0.028; 95% CI = − 33.2 to − 2.5); executive function (β = − 9.8, p = 0.08; 95% CI = − 21 to 1)] and gross motor development (β = − 13; p = 0.022; 95% CI = − 24 to − 2), whereas in boys cortisol levels were negatively associated with socioemotional development [autistic-like behaviours: Incidence Rate Ratio (IRR) = 1.6, p = 0.039; 95% CI = 1.01 to 2.41]. Salivary alpha-amylase was positively associated with socioemotional development in boys only [social competence (β = 2.11, p = 0.013; 95% CI = 0.47 to 3.72), autistic-like behaviours (IRR = 0.93, p = 0.042; 95% CI = 0.87 to 0.99) and hyperactivity symptoms (IRR = 0.81, p = 0.021; 95% CI = 0.69 to 0.97)]. These results suggest that stress system activity in early life is associated with longer-term neurodevelopment and that sex is an important factor in this relationship.