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The Premature Infant Pain Profile (PIPP) is a promising multidimensional tool for comparison and testing of new technologies in newborn pain assessment studies since it may adhere to basic psychophysical parameters of intensity, direction, reactivity, regulation and slope described in analyses of physiological pain indicators. The aim of this study was to evaluate whether these psychophysical parameters can be achieved using the PIPP in acute pain assessment. Thirty-six healthy term newborn infants were conveniently sampled whilst being videotaped before, during and after heel prick blood sampling. The images were blind-scored by three trained independent raters and scored against the PIPP. The PIPP and its facial action indicators met the parameters of intensity, reactivity and regulation (all p < 0.001). The heart rate variability did not meet any parameter (all p > 0.05). The oxygen saturation variability met only the intensity parameter (p < 0.05). The behavioural state indicator met all parameters and had the best correlation to the psychophysical parameters of all indicators of PIPP (all p < 0.001). We concluded that the overall PIPP meets the assumptions of these psychophysical parameters, being the behavioural state indicator which best fit the model.