Betamethasone is widely used to enhance fetal lung maturation in case of threatened preterm birth. Antenatal corticosteroids are known to reduce fetal heart rate variability (fHRV) in the days following administration. Since decreased fHRV is a marker for fetal distress, this transient decrease of fHRV can cause unnecessary medical intervention. To describe the effect of betamethasone on fHRV, by applying spectral analysis on non-invasive fetal electrocardiogram (fECG) recordings. Secondary analysis of a prospective cohort study. Women with a singleton pregnancy, at risk for preterm delivery and receiving betamethasone, admitted to the obstetric high care unit in the period from March 2013 until July 2016. The primary outcome measure was fHRV in both time- and frequency-domain. Secondary outcome measures included basal fetal heart rate (fHR) and fHR variance. FHRV parameters were then calculated separately for the quiet and active state. Following 68 inclusions, 22 patients remained with complete series of measurements and sufficient data quality. FHRV parameters and fHR showed a decrease on day 2 compared to day 1, significant for short-term variability and high-frequency power. Similar results were found when analyzing for separate behavioral states. The number of segments in quiet state increased during days 1 and 2. Normalized values showed no difference for all behavioral states. FHRV decreases on day 2 after betamethasone administration, while periods of fetal quiescence increase. No changes were found in the normalized values, indicating that the influence of autonomic modulation is minor. Clinical trial registration number NL43294.015.13.