Table 1.

Indicators of neonatal pain.

Category	Description
Behavioural signs
Alertness and sleep-wake state	A hyper-alert state is often seen during on-going or prolonged pain Younger or sicker infants can need longer time to comfort themselves and return to rest after a painful event
Body movements and muscle tension	Full-term infants withdraw limbs from pain, whereas in preterm infants these responses are more diffuse and harder to control Strong infants can react with increased muscle tension in arms and legs Younger or sicker infants can turn flaccid
Crying	Healthy, awake, and full-term newborn infants react with moaning or crying that gets more intense and with longer duration with increasing discomfort. In contrast, infants of lower gestational age, sick, in a sleeping state, or sedated will have difficulties showing a vocal reaction so the absence of crying must not be taken for absence of pain; crying can often be noted as a weaker moaning sound
Facial activity, grimacing	Most used facial expressions that are indicators of pain: ○Brow bulge ○Squeezed eyes ○Distinct furrows from the nose to the ends of the mouth ○Tense and stretched mouth and tongue ○Raised cheeks
Physiological signs
Heart rate and heart rate variability (HRV)	In healthy full-term infants, pain triggers an increased heart rate which leads to increased blood pressure and a red skin color Conversely, younger and sicker infants may react with an increase or decrease in heart rate, possibly bradycardia Pain reduces HRV HRV is best suited for assessing prolonged pain
Respiration rate	In robust infants respiration rate will increase as a result of acute pain, whereas the opposite, and even apneas can be seen in preterm infants who do not have energy to trigger the fight-or-flight system Pain in the thorax-region can lead to impaired breathing
Oxygen saturation	Changes in oxygen saturation follows pain-associated changes in respiration and heart rate Both an increase and decrease of oxygen saturation can be seen, depending on the context
Cortisol	Increased levels can be seen after surgery [35] and painful procedures [36] Measured in plasma, saliva, or urine High variability, more useful in research than clinically
Neurophysiological signs
Cerebral oxygenation	Pain measured with Near Infrared Spectroscopy (NIRS) associated with cerebral activation and changes in oxygenated and deoxygenated haemoglobin concentration in the brain An increase in oxygenated haemoglobin is assumed to reflect neuronal activation caused by pain NIRS is sensitive to movement artefacts [37,38]
EEG, evoked potentials	Multi-channel EEG used as a proxy for neuronal activity with the majority of studies reporting on pain-related event-related potentials during acute painful procedures [39,40] Primarily used in research, no standard clinical pain assessment method/tool
Functional magnetic resonance imaging (fMRI)	Has shown which areas in the infant brain are activated by pain [41,42] Not relevant as a bedside pain assessment tool Can help in developing and validating other measures