The level of circulating vitamin D is known to be associated with the ejection fraction in heart failure patients and studies in rats have shown that vitamin D depletion leads to increased levels of circulating norepinephrine and decreased atrial contractility. We elucidated the effects of vitamin D supplementation on the autonomous nervous system in healthy youngsters. Thirty healthy subjects aged 18-25 years were recruited based on their serum 25-hydroxyvitamin D (25[OH]D): 15 vitamin D insufficient (25[OH]D < 50 nmol/L) and 15 vitamin D sufficient (25[OH]D > 80 nmol/L) subjects. Both groups had vitamin D supplementation (30 microg/day) and were tested at baseline and after 30, 90, and 180 days. At each visit the serum 25-hydroxyvitamin D was measured and the head-up tilt test performed. Serum 25[OH]D remained stable in the vitamin D sufficient group, while the insufficient group had a significant increase (32.0-64.5 nmol/L), P < 0.0001. Despite the increase, the insufficient group did not reach the level of the vitamin D sufficient group within the 6 months observational period (96.1 vs 64.5 nmol/L), P < 0.01. Serum norepinephrine at baseline was higher in the insufficient group (mean = 1.61 nmol/L) than in the vitamin D sufficient group (mean = 0.94 nmol/L), P < 0.01, whereas the response to tilt was lower in the insufficient group (mean = 0.69 nmol/L) compared to the sufficient group (mean = 1.17 nmol/L), P < 0.01. The heart rate at rest was higher in the insufficient group (mean = 67.7 bpm) than in the vitamin D sufficient group (mean = 56.6 bpm), P < 0.01, for the three first visits. At the last visit no difference was found. The systolic and diastolic blood pressure differed between the groups after a month, with higher pressures in the insufficient group than in the sufficient group. Vitamin D supplementation modulates the sympathetic nervous system in healthy youngsters with low serum vitamin D. The observation might lead to a greater focus on possible prevention of cardiac disease later on in life by vitamin D supplementation early in life.