Many hormones are released in a pulsatile or burst-like pattern resulting in fluctuating blood levels that can undergo rapid modulation by physiological and pathological signals. To accurately measure these changes in hormone concentration requires frequent blood sampling, often over extended periods as the overall rhythmicity may vary over 24 hours. The aim of this study was to develop a computerized, automated blood sampling system which allows repeated stress-free blood sample collection from humans over an extended period under basal or test conditions. The system incorporates a peristaltic pump, fraction collector and standard infusion pump together with a custom built electronic control unit linked to a personal computer. Disposable tubing prevents cross-contamination between study participants. The computer programme is modifiable to adjust for the number of specimen tubes and volume of blood collected per sampling cycle. Patency of the collecting line is maintained with 0.9% saline, without the need for heparinization. To validate the system, 10-minute samples for cortisol were collected over 24 hours from five healthy volunteers, of whom two had additional concomitant ACTH sampling. Deconvolution analysis revealed an expected number of hormone secretory episodes and a non-pathological degree of orderliness within the data. There was high concordance between ACTH and cortisol secretory events. The ability of the system to allow multiple measurements and of the software program to link with other physiological monitoring equipment provides a powerful tool to study physiologic/pathophysiologic change in relation to blood hormone and other biomarker levels.