BACKGROUND: Pain in the neonate is associated with acute behavioural and physiological changes. Cumulative pain is associated with morbidities, including adverse neurodevelopmental outcomes. Studies have shown a reduction in changes in physiological parameters and pain score measurements following pre-emptive analgesic administration in neonates experiencing pain or stress. Non-pharmacological measures (such as holding, swaddling and breastfeeding) and pharmacological measures (such as acetaminophen, sucrose and opioids) have been used for analgesia. This is an update of a review first published in 2006 and updated in 2012. OBJECTIVES: The primary objective was to evaluate the effectiveness of breastfeeding or supplemental breast milk in reducing procedural pain in neonates. The secondary objective was to conduct subgroup analyses based on the type of control intervention, gestational age and the amount of supplemental breast milk given. SEARCH METHODS: We searched CENTRAL, MEDLINE, Embase, CINAHL and trial registries (ICTRP, ISRCTN and clinicaltrials.gov) in August 2022; searches were limited from 2011 forwards. We checked the reference lists of included studies and relevant systematic reviews. SELECTION CRITERIA: We included randomised controlled trials (RCTs) or quasi-RCTs of breastfeeding or supplemental breast milk versus no treatment/other measures in neonates. We included both term (≥ 37 completed weeks postmenstrual age) and preterm infants (< 37 completed weeks' postmenstrual age) up to a maximum of 44 weeks' postmenstrual age. The study must have reported on either physiological markers of pain or validated pain scores. DATA COLLECTION AND ANALYSIS: We assessed the methodological quality of the trials using the information provided in the studies and by personal communication with the authors. We extracted data on relevant outcomes, estimated the effect size and reported this as a mean difference (MD). We used the GRADE approach to assess the certainty of evidence. MAIN RESULTS: Of the 66 included studies, 36 evaluated breastfeeding, 29 evaluated supplemental breast milk and one study compared them against each other. The procedures conducted in the studies were: heel lance (39), venipuncture (11), intramuscular vaccination (nine), eye examination for retinopathy of prematurity (four), suctioning (four) and adhesive tape removal as procedure (one). We noted marked heterogeneity in the control interventions and pain assessment measures amongst the studies. Since many studies included multiple arms with breastfeeding/supplemental breast milk as the main comparator, we were not able to synthesise all interventions together. Individual interventions are compared to breastfeeding/supplemental breast milk and reported. The numbers of studies/participants presented with the findings are not taken from pooled analyses (as is usual in Cochrane Reviews), but are the overall totals in each comparison. Overall, the included studies were at low risk of bias except for masking of intervention and outcome assessment, where nearly one-third of studies were at high risk of bias. Breastfeeding versus control Breastfeeding may reduce the increase in heart rate compared to holding by mother, skin-to-skin contact, bottle feeding mother's milk, moderate concentration of sucrose/glucose (20% to 33%) with skin-to-skin contact (low-certainty evidence, 8 studies, 784 participants). Breastfeeding likely reduces the duration of crying compared to no intervention, lying on table, rocking, heel warming, holding by mother, skin-to-skin contact, bottle feeding mother's milk and moderate concentration of glucose (moderate-certainty evidence, 16 studies, 1866 participants). Breastfeeding may reduce percentage time crying compared to holding by mother, skin-to-skin contact, bottle feeding mother's milk, moderate concentration sucrose and moderate concentration of sucrose with skin-to-skin contact (low-certainty evidence, 4 studies, 359 participants). Breastfeeding likely reduces the Neonatal Infant Pain Scale (NIPS) score compared to no intervention, holding by mother, heel warming, music, EMLA cream, moderate glucose concentration, swaddling, swaddling and holding (moderate-certainty evidence, 12 studies, 1432 participants). Breastfeeding may reduce the Neonatal Facial Coding System (NFCS) score compared to no intervention, holding, pacifier and moderate concentration of glucose (low-certainty evidence, 2 studies, 235 participants). Breastfeeding may reduce the Douleur Aigue Nouveau-né (DAN) score compared to positioning, holding or placebo (low-certainty evidence, 4 studies, 709 participants). In the majority of the other comparisons there was little or no difference between the breastfeeding and control group in any of the outcome measures. Supplemental breast milk versus control Supplemental breast milk may reduce the increase in heart rate compared to water or no intervention (low-certainty evidence, 5 studies, 336 participants). Supplemental breast milk likely reduces the duration of crying compared to positioning, massage or placebo (moderate-certainty evidence, 11 studies, 1283 participants). Supplemental breast milk results in little or no difference in percentage time crying compared to placebo or glycine (low-certainty evidence, 1 study, 70 participants). Supplemental breast milk results in little or no difference in NIPS score compared to no intervention, pacifier, moderate concentration of sucrose, eye drops, gentle touch and verbal comfort, and breast milk odour and verbal comfort (low-certainty evidence, 3 studies, 291 participants). Supplemental breast milk may reduce NFCS score compared to glycine (overall low-certainty evidence, 1 study, 40 participants). DAN scores were lower when compared to massage and water; no different when compared to no intervention, EMLA and moderate concentration of sucrose; and higher when compared to rocking or pacifier (low-certainty evidence, 2 studies, 224 participants). Due to the high number of comparator interventions, other measures of pain were assessed in a very small number of studies in both comparisons, rendering the evidence of low certainty. The majority of studies did not report on adverse events, considering the benign nature of the intervention. Those that reported on adverse events identified none in any participants. Subgroup analyses were not conducted due to the small number of studies. AUTHORS' CONCLUSIONS: Moderate-/low-certainty evidence suggests that breastfeeding or supplemental breast milk may reduce pain in neonates undergoing painful procedures compared to no intervention/positioning/holding or placebo or non-pharmacological interventions. Low-certainty evidence suggests that moderate concentration (20% to 33%) glucose/sucrose may lead to little or no difference in reducing pain compared to breastfeeding. The effectiveness of breast milk for painful procedures should be studied in the preterm population, as there are currently a limited number of studies that have assessed its effectiveness in this population.