Evidence is lacking as to whether ambient light or phototherapy light could interfere with pulse oximeter performance. In this randomised cross-over trial, we recruited neonates of gestation >24 weeks. Consented infants were randomly assigned to either pulse oximeter sensor with opaque wrap or without opaque wrap. Nellcor and Masimo sensors were applied simultaneously to different feet for 10 min of recording. Infants were crossed over to the other intervention for a further 10 min, totalling 20 min recording per infant. Primary outcome was faster acquisition of data with shielding of pulse oximeter sensor as compared with not shielding. A total of 96 babies were recruited. There was no difference in primary outcome of time taken to display valid data between the two groups (opaque wrap: 12.73±3.1 s vs no opaque wrap: 13.16±3.3 s, p=0.27). There was no difference in any of the secondary outcomes (percentage of valid data points, percentage of time saturation below target, and so on) between the two groups in both pulse oximeters. Masimo sensor readings displayed a higher mean oxygen saturation (mean difference of 2.85, p=0.001) and lower percentage of time saturation below 94% (mean difference of -27.8, p=0.001) than Nellcor in both groups. There was no difference in any of the outcomes in babies receiving phototherapy (n=21). In this study, shielding the pulse oximeter sensor from ambient light or phototherapy light did not yield faster data acquisition or better data quality. ISRCTN10302534. © Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.