The purpose of this randomized crossover trial was to determine the effects on autonomic responses in preterm infants of longer Kangaroo Care (30 minutes, KC30) and shorter KC (15 minutes, KC15) before and throughout heel stick compared with incubator care (IC). Beat-to-beat heart rate (HR) and spectral power analysis of heart rate variability, low frequency power (LF), high frequency power (HF), and LF/HF ratio were measured in 26 infants. HR changes from Baseline to Heel Stick were significantly less in KC30 and KC15 than in IC, and more infants had HR decrease in IC than in 2 KC conditions. In IC, LF and HF significantly increased from Baseline to Heel Stick and dropped from Heel Stick to Recovery; in 2 KC conditions, no changes across study phases were found. During Heel Stick, LF and HF were significantly higher in IC than in KC30. In all 3 conditions, LF/HF ratio decreased from Baseline to Heel Stick and increased to Recovery; no differences were found between IC and two KC conditions. Both longer and shorter KC before and throughout heel stick can stabilize HR response in preterm infants, and longer KC significantly affected infants' sympathetic and parasympathetic responses during heel stick compared with incubator care. PERSPECTIVE This study showed that KC has a significant effect on reducing autonomic pain responses in preterm infants. The findings support that KC is a safe and effective pain intervention in the neonatal intensive care unit.