Abstract Background Gender-specific differences in hypothalamus–pituitary–adrenal (HPA) axis activity have been postulated to emerge during puberty. We conducted a systematic review and meta-analysis to test the hypothesis that gender-specific differences in HPA axis activity are already present in childhood. Methods From inception to January 2016, PubMed and EMBASE.com were searched for studies that assessed non-stimulated cortisol in serum or saliva or cortisol in 24-h urine in healthy males and females aged ≤18 years. Studies that conform with the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) statement were reported. Standardized mean differences (95% CIs) were calculated and analyzed using fixed-effect meta-analysis stratified for age: