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Objective Providing Kangaroo care (K-care) helps comfort infants through painful and stressful procedures including retinopathy screening examination (RSE) for retinopathy of prematurity. We examined the early pain-related and delayed inflammatory effects of K-care during the first RSE in preterm infants.Design Randomised clinical trial.Setting In a family-centred neonatal intensive care unit between 1 March 2023 and 31 August 2024.Patients Preterm infants (gestation <34+0 weeks and/or birth weight <2000 g) who required RSEs.Interventions Patients were randomly assigned to K-care provided by mother or father during RSE or control group on an examination table (n=50/group).Main outcome measures The Premature Infant Pain Profile (PIPP) score within the first minute of RSE was measured. Salivary samples (>110 uL) were collected before, at 1 and 6–8 hours after RSE for assays of cortisol, interleukin-1β, interleukin-6 and tumour necrosis factor-alpha (TNF-α) levels by ELISA methods in a blinded fashion.Results 100 infants (mean±SD gestational age: 31.1±2.4 weeks; birth weight: 1496±452 g, 58% males) were enrolled. Compared with controls (n=50), infants of the K-care group (n=50) had significantly lower PIPP score (11.0 vs 12.9; p=0.002); salivary cortisol levels (4.9 vs 6.9 ng/mL; p=0.001) at 1 hour after RSE, interleukin-6 (31.9 vs 34.7 pg/mL; p=0.01) and TNF-α (53.9 vs 58.6 pg/mL; p=0.02), but not interleukin-1β (50.4 vs 53.0 pg/mL; p=0.46), levels at 6–8 hour after RSE.Conclusions Providing K-care during RSE in preterm infants may potentially reduce pain-related stress, cortisol and inflammatory responses.