Major depressive disorder (MDD) has been consistently associated with hypothalamic-pituitary-adrenal (HPA)-axis and autonomic nervous system (ANS) (re-)activity abnormalities, however, often with conflicting results.This study offers a concurrent multi-measure assessment of both HPA-axis and ANS activity and reactivity over 3 days to better characterize baseline and dynamic neuroendocrine alterations in MDD accounting for multiple individual factors. We therefore investigated group differences between 20 unmedicated MDD patients and 20 carefully-matched healthy controls (HC) by simultaneously assessing morning plasma (CORT, ACTH, copeptin) and awakening response saliva (CORT, DHEA, DHEA-s) endocrine measures, as well as multiple linear and non-linear measures of resting heart rate (HR) and its variability (HRV), before (baseline, day 1) and after a successive overnight metyrapone (HPA-axis stimulation, day 2) and dexamethasone (HPA-axis suppression, day 3) pharmaco-endocrine challenge, controlling for childhood trauma (CT) history.Statistically significant group differences emerged only for baseline plasma CORT and ACTH levels (MDD > HC) and resting HR in all 3 days. No differences were found in dynamic plasma levels and all saliva endocrine measures, as well as all HRV measures. Baseline HR was the only significant predictor for MDD diagnosis.Our detailed baseline and dynamic neuroendocrine comparison using closely matched HC indicates fewer neuroendocrine alterations in MDD than expected. These results challenge prior findings and support the importance of exact matching when investigating neuroendocrine biomarkers, as previously reported findings may rely on unaccounted individual but not group differences.