Measures of HR and HRV offer multiple indices of reactivity to painful events. These measures are particularly helpful in preterm and ill infants where distress signals are often nonspecific and ambiguous. HR is easy to acquire, and a variety of widely used techniques are available for processing it. In general, the neuroanatomic and neurophysiologic bases for pain perception are in place even in the most preterm infant and produce patterns of HR and HRV responses that are similar across multiple settings. Developmental and experiential factors related to preterm birth, however, may affect these HR responses. Furthermore, evaluation of ill infants in an NICU setting adds multiple contextual factors that potentially influence HR and HRV and alter their specificity as measures of pain. In some cases, it may appear that pain reactivity is reduced when, in fact, HR reactivity is only an expression of the biologic capacity to produce a response, not the presence of a response itself. The nature of the setting and the infant's health, developmental stage, and behavioral state all contribute to potentially altering HR responses to painful events in this setting. Thus, the methodology used and its application must be flexible. A variety of HRV analysis techniques may be needed to identify a variety of response patterns and mechanisms that influence pain reactivity. Furthermore, careful selection of HR epochs for stationarity, an understanding of the potential discordance between biologic and behavioral measures, the effects of medication, and an accounting for developmental differences that occur during a typical NICU course are all critical factors for investigators to be aware of. Understanding cardiovascular reactivity as a measure of response to painful events in vulnerable infants requires ongoing work.