New Findings r What is the central question of this study? Can power spectral analysis of fetal heart rate variability provide a reliable, objective estimate of autonomic balance before birth? r What is the main finding and its importance? This study demonstrates that there is a substantial increase in fetal heart rate spectral power over late gestation in chronically instrumented fetal sheep, with changes between sleep states that are consistent with known patterns of autonomic activity. However, parasympathetic blockade with atropine and complete chemical sympathectomy did not support substantial, consistent differences in the frequencies of sympathetic and parasympathetic activity in late gestation, suggesting that this does not provide a reliable index of relative cardiac autonomic activity before birth. Power spectral analysis of fetal heart rate variability has been proposed to provide a non-invasive estimate of autonomic balance. However, there are few systematic data before birth. We therefore examined developmental changes in the frequency power spectrum at very low (0-0.04 Hz), low (0.04-0.15 Hz) and high frequencies (0.15-0.4 Hz), as well as the ratio of low-to high-frequency power (LF/HF), in chronically catheterized, healthy fetal sheep at 0.6 (n = 8), 0.7 (n = 7) and 0.8 gestational age (ga; n = 11). In a second study, 0.8 ga fetuses received either atropine (4.8 mg bolus, then 4.8 mg h −1 for 30 min, n = 6) or 6-hydroxydopamine (20 mg ml −1 at 2.5 ml h −1 for 3 h; n = 9). Data were analysed by sleep state, defined by low-voltage-high-frequency (LV) or high-voltage-low-frequency (HV) EEG. Total spectral power increased with gestational age (P < 0.05), while LF/HF decreased from 0.6 to 0.7 ga. At 0.8 ga, heart rate and LF/HF were significantly higher during HV than LV sleep (P < 0.05). Consistent with this, although total spectral power was not significantly greater during HV sleep, there was a significant interaction between sleep state and frequency band (P = 0.02). Both atropine (P = 0.05) and 6-hydroxydopamine (P < 0.05) were associated with an overall reduction in spectral power but no significant effect on the LF/HF ratio. This study does not support substantial, consistent differences between the frequencies of sympathetic and parasympathetic activity in late-gestation fetal sheep.