Our immediate goal was to study heart rate variability (HRV) in chicken embryos in the egg. Instantaneous heart rate data were needed for this purpose, and accordingly an ECG recording method in the egg was developed. The aim of this work was to test the hypothesis that autonomic nervous cardiac modulation, as shown from HRV parameters, is present at the end of development and that it reaches a constant value during the last days of incubation. Embryonic chicken heart rate was obtained at the final incubation period (days 19 and 20) from ECG recordings. Tachograms were computed and time- and frequency-domain indices of HRV were determined. No significant differences were found between HRV indices from day 19 and day 20. The power spectra extended in two frequency bands with centre frequency around 0.6-0.7 Hz (low frequency (LF) component), and another around 1.2-1.5 Hz (high frequency (HF) component); the latter was shown to reflect respiratory sinus arrhythmia. A relation between mean RR interval and some HRV parameters (rMSSD, pNN5 and HF power) was shown. HRV results obtained from embryonic chickens, showed the presence of modulation of cardiovascular function by the autonomic nervous system. The results suggested that sympathetic and parasympathetic activities have already reached a constant level at day 19 of incubation. High frequency oscillations (0.78-2.5 Hz) were detected and are considered to reflect respiratory sinus arrhythmia.