The aim of the investigation was to assess pain by frequency domain analysis of heart rate variability (HRV) during a routine heel lancing procedure in term new-born infants. Beat-to-beat heart rate (HR) was recorded in 23 healthy new-born infants on the maternity ward during blood sampling for neonatal screening. A sham heel prick prior to the sharp lancing procedure was performed randomly in half of the infants. Spectral analysis of HRV was assessed for each of the following sequences: (1) baseline (2) sham heel prick (3) sharp heel prick and (4) squeezing the heel for blood sampling. The response to the sham prick did not differ significantly from the sharp prick. Compared with the baseline, sharp lancing gave rise to minor increases in HR and variability in the low frequency band of the spectral analysis. A clear stress response was provoked when the heel was squeezed for blood sampling, indicated by an increased HR and a decreased spectral power in the high frequency band (i.e. lower vagal tone). The different stress responses during the lancing and the squeezing of the heel were clearly illustrated when principal component analysis was applied and the vectors for the changes in HR and spectral pattern were indicated. In conclusion, the squeezing of the heel is the most stressful event during the heel prick procedure.