Of all malignancies in children, acute lymphoblastic leukemia (ALL) is the most common type. Since survival significantly improves over time, treatment-related side effects become increasingly important. Glucocorticoids play an important role in the treatment of ALL, but they may suppress the hypothalamic-pituitary-adrenal (HPA) axis. The duration of HPA axis suppression is not yet well defined. The present study aimed at assessing the function of the HPA axis by determining the cortisol awakening response (CAR) and the dexamethasone (DEX) suppression test in children that were treated for childhood ALL, compared to a healthy age and sex matched reference group. In addition, questionnaires regarding sleep, fatigue, depression and quality of life were completed by the children and their parents. Fourty-three survivors who finished their treatment for childhood ALL 37 (interquartile range 22-75) months before and 57 healthy controls were included. No differences in CAR were observed between ALL survivors and the reference group, but survivors of ALL had higher morning cortisol levels and an increased cortisol suppression in response to oral dexamethasone. Higher cortisol levels in childhood ALL survivors were associated with more fatigue and poorer quality of life. We conclude that the experience of a stressful life event in the past may have caused a long-term dysregulation of the HPA axis in childhood ALL survivors, as reflected in an increased cortisol production and an enhanced negative feedback mechanism.