Beneficial effects of antenatal glucocorticoid treatment in pregnancies at risk for preterm delivery may entail long-term consequences for the establishment of sympathoadrenergic system balance. We analyzed the cardiac autonomic system activity in neonates with a single course of antenatal betamethasone (2 x 12 mg) treatment by calculating heart rate variability (HRV) time-domain parameters from 24 h ECG recordings and short-term frequency-domain parameters during infant active and resting states. In addition, resting and challenged salivary alpha-amylase levels were measured in 23 betamethasone-exposed neonates and compared with controls. Indicators for overall HRV (SDNN: p = 0.258; triangular index: p = 0.179) and sympathovagal balance [low- to high-frequency power (LF/HF): p = 0.82 (resting state)] were not significantly different in neonates of the betamethasone treatment group. Parameters mostly influenced by sympathetic activity [SD of the average of valid NN intervals (SDANN): p = 0.184 and SDs of all NN intervals (SDNNi): p = 0.784] and vagal tone [RMSSD: p = 1.0; NN50: p = 0.852; HF: p = 0.785 (resting state)] were unaltered. Resting alpha-amylase levels were not significantly different in the betamethasone treatment group (p = 0.304); however, alpha-amylase release after a neonatal challenge was slightly reduced (p = 0.045). Thus, cardiac autonomic balance seems to be preserved in neonates exposed to a single course of antenatal betamethasone treatment.