Multiple researchers have validated indicators and measures of infant pain. However, infants at risk for neurologic impairment (NI) have been under studied. Therefore, whether their pain responses are similar to those of other infants is unknown. Pain responses to heel lance from 149 neonates (GA>25-40 weeks) from 3 Canadian Neonatal Intensive Care units at high (Cohort A, n=54), moderate (Cohort B, n=45) and low (Cohort C, n=50) risk for NI were compared in a prospective observational cohort study. A significant Cohort by Phase interaction for total facial action (F(6,409)=3.50, p=0.0022) and 4 individual facial actions existed; with Cohort C demonstrating the most facial action. A significant Phase effect existed for increased maximum Heart Rate (F(3,431)=58.1, p=0.001), minimum Heart Rate (F(3,431)=78.7, p=0.001), maximum Oxygen saturation (F(3,425)=47.6, p=0.001), and minimum oxygen saturation (F(3,425)=12.2, p=0.001) with no Cohort differences. Cohort B had significantly higher minimum (F(2,79)=3.71, p=0.029), and mean (F(2,79)=4.04, p=0.021) fundamental cry frequencies. A significant Phase effect for low/high frequency Heart Rate Variability (HRV) ratio (F(2,216)=4.97, p=0.008) was found with the greatest decrease in Cohort A. Significant Cohort by Phase interactions existed for low and high frequency HRV. All infants responded to the most painful phase of the heel lance; however, infants at moderate and highest risk for NI exhibited decreased responses in some indicators.