Heart rate and RR variation (the standard deviation of the mean RR interval for a 5-min period) were evaluated as measurements of cardiac parasympathetic nervous system activity in fasting supine diabetic (N = 22) and comparable age normal (N = 22) subjects. The rate of breathing did not effect heart rate, but was inversely related to the RR variation (r = 0.89, P < 0.01). Heart rate was increased (P < 0.0001) and RR variation decreased (P < 0.05) during β-adrenergic stimulation with isoproterenol and during parasympathetic blockade with atropine (both P < 0.0001). Hence, the cardiac effects of β-adrenergic stimulation may mimic the effects of diminished parasympathetic function. To evaluate parasympathetic control of RR variation, independently of possible effects of increased sympathetic activities, studies were performed during β-adrenergic blockade with propranolol. RR variation during propranolol was less both in 14 diabetic subjects without clinical symptoms of autonomic neuropathy (P < 0.005) and in 8 diabetics with clinical symptoms of autonomic neuropathy (P < 0.001) when compared with 22 age-comparable normal subjects. The measurement of RR variation was very reproducible with a day-to-day coefficient of variation of 9.7 ± 2.8﹪ (x̄ ± SEM) in diabetic subjects with stable hyperglycemia. It is concluded that supine RR variation during a deep respiratory rate and during β-adrenergic blockade is a sensitive, quantitative, and reproducible method to evaluate parasympathetic nervous activity in normal and diabetic subjects. Furthermore, cardiac parasympathetic activity may be diminished in diabetic subjects before clinical symptoms of autonomic neuropathy are evident.