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Objective Non-invasive spectral analysis of fetal heart rate variability is a promising new field of fetal monitoring. To validate this method properly, we studied the relationship between gestational age and the influence of fetal rest–activity state on spectral estimates of fetal heart rate variability. Design Prospective longitudinal study. Setting Tertiary care teaching hospital. Population Forty healthy women with an uneventful singleton pregnancy. Methods Non-invasive fetal electrocardiogram measurements via the maternal abdomen were performed at regular intervals between 14 and 40 weeks of gestation and processed to detect beat-to-beat fetal heart rate. Simultaneous ultrasound recordings were performed to assess fetal rest–activity state. Main outcome measures Absolute and normalized power of fetal heart rate variability in the low (0.04–0.15 Hz) and high (0.4–1.5 Hz) frequency band were obtained, using Fourier Transform. Results 14% of all measurements and 3% of the total amount of abdominal data (330 segments) was usable for spectral analysis. During 21–30 weeks of gestation, a significant increase in absolute low and high frequency power was observed. During the active state near term, absolute and normalized low frequency power were significantly higher and normalized high frequency power was significantly lower compared with the quiet state. Conclusions The observed increase in absolute spectral estimates in preterm fetuses was probably due to increased sympathetic and parasympathetic modulation and might be a sign of autonomic development. Further improvements in signal processing are needed before this new method of fetal monitoring can be introduced in clinical practice.