Rhythmicity is a intrinsic feature of biological systems, including the hypothalamic-pituitary-adrenal axis, a mammalian neurohormonal system crucial both in daily life and as a network that responds to stressful stimuli. Circadian and ultradian rhythmicity underlie HPA activity in rodents and in humans, regulating gene expression, metabolism and behavior, and adverse consequences occur when rhythms are disturbed. In the assessment of human disease, the complexity of HPA rhythmicity is rarely acknowledged or understood, and is currently a limitation to better diagnosis and treatment. However, the recent emergence of ambulatory, high frequency and blood-free hormone sampling techniques has the promise to substantially change our understanding of the function of HPA axis in healthy normal life, and provide new opportunities for the diagnosis and treatment of disease.