Diurnal variation of baseline startle amplitude was examined in 14 normal inpatients on a research unit where behavioral activity and environmental stimuli were highly controlled. We tested a hypothesized association between diurnal variations of salivary cortisol and reflex amplitude by recording acoustic startle eyeblinks shortly before bedtime, when cortisol was near its lowest daily level, and just after awakening, when cortisol was at its peak. Results showed that startle eyeblinks were greater during evening than morning sessions, whereas the opposite was true for cortisol levels. Skin conductance levels and reaction time performance also increased from morning to evening. These findings are consistent with accumulating evidence suggesting a possible link between startle reactivity and hypothalamic-pituitary-adrenal axis activity, and an association between diurnal variations in endogenous arousal and startle amplitude.