Analysis of heart rate variability (HRV) is a recognized tool in the assessment of autonomic nervous system (ANS) activity. Indeed, both time and spectral analysis techniques enable us to obtain indexes that are related to the way the ANS regulates the heart rate. However, these techniques are limited in terms of the lack of thresholds of the numerical indexes, which is primarily due to high inter-subject variability. We proposed a new fetal HRV analysis method related to the parasympathetic activity of the ANS. The aim of this study was to evaluate the performance of our method compared to commonly used HRV analysis, with regard to i) the ability to detect changes in ANS activity and ii) inter-subject variability. This study was performed in seven sheep fetuses. In order to evaluate the sensitivity and specificity of our index in evaluating parasympathetic activity, we directly administered 2.5 mg intravenous atropine, to inhibit parasympathetic tone, and 5 mg propranolol to block sympathetic activity. Our index, as well as time analysis (root mean square of the successive differences; RMSSD) and spectral analysis (high frequency (HF) and low frequency (LF) spectral components obtained via fast Fourier transform), were measured before and after injection. Inter-subject variability was estimated by the coefficient of variance (%CV). In order to evaluate the ability of HRV parameters to detect fetal parasympathetic decrease, we also estimated the effect size for each HRV parameter before and after injections. As expected, our index, the HF spectral component, and the RMSSD were reduced after the atropine injection. Moreover, our index presented a higher effect size. The %CV was far lower for our index than for RMSSD, HF, and LF. Although LF decreased after propranolol administration, fetal stress index, RMSSD, and HF were not significantly different, confirming the fact that those indexes are specific to the parasympathetic nervous system. In conclusion, our method appeared to be effective in detecting parasympathetic inhibition. Moreover, inter-subject variability was much lower, and effect size higher, with our method compared to other HRV analysis methods.