Acute stress response measures serve as an indicator of physiological functioning, but have previously led to contradictory results in young children due to age-related cortisol hypo-responsivity and methodological inconsistencies in assessment. The aim of this study was to investigate stress responses during a validated age-adapted socio-evaluative stress task in children aged 2-6 years in a child care environment and to detect socio-demographic, task- and child-related characteristics of stress responses. Stress responses were assessed in 323 children for salivary cortisol and salivary alpha amylase (sAA), and in 328 children for changes in heart rate variability (HRV). These data were then associated with socio-demographic (e.g. SES), task-related (e.g. task length) and child-related characteristics (e.g. self-regulation) of stress responses using multilevel models. Analyses revealed elevated sympathetic reactivity (sAA: Coeff=0.053, p=0.004) and reduced HRV (Coeff=-0.465, p<0.001), but no hypothalamic-pituitary-adrenal (HPA) response (Coeff=0.017, p=0.08) during the stress task. Child's age (Coeff=-5.82, p<0.001) and movement during the task (Coeff=-0.17, p=0.015) were associated with acute cortisol release, while diurnal sAA was associated with acute sAA release (Coeff=0.24, p<0.001). Age (Coeff=-0.15, p=0.006) and duration of the task (Coeff=0.13, p=0.015) were further associated with change of HRV under acute stress condition. Children showed inconsistent stress responses which contradicts the assumption of a parallel activation of both stress systems in a valid stress task for young children and might be explained by a pre-arousal to the task of young children in a child care setting. Further results confirm that child- and task-related conditions need to be considered when assessing stress responses in these young children.