Throughout the duration of the test, babies were continuously recorded with a video camera at least 2 minutes before sampling and 2 minutes after the procedure. The heel was warmed up by a glove with lukewarm water at least 2 minutes before the sample. Heel lance was made with an automated piercing device for routine neonatal screening for congenital disorders at 48 hours of life. Heart rate was monitored by a pulse oximeter (Radical MasimoSet Datascope, Masimo Corporation, Irvine, CA) set on the infant's hand or foot. Hear rate was measured continuously but special attention was given to three time points: t0 (2 minutes before sampling); t1 (the highest value of the first 10 seconds after heel prick); and t2 (2 minutes after the procedure).