BACKGROUND/AIMS: Retinopathy of prematurity (ROP) screening is vital for early disease detection in very premature infants but can cause physiological instability. This study compares the physiological response to binocular indirect ophthalmoscopy (BIO) with indentation and non-contact ultra-widefield (UWF) retinal imaging in non-ventilated neonates. The impact of the Dandle WRAP, a specialised swaddling aid, on UWF imaging was also assessed. METHODS: This retrospective study included 86 ROP screening events in 66 non-ventilated infants aged 35.3 weeks (range 30.6-44.6). Vital signs were continuously recorded, evaluating immediate (within 15 min) and longer-term (within 12 h) physiological responses. RESULTS: ROP screening significantly increased heart and respiratory rates and decreased oxygen saturation within 15 min of screening. No significant differences in physiological responses were found between BIO and UWF imaging, although there was a trend towards lower maximum heart rate with UWF imaging. The Dandle WRAP did not significantly alter physiological responses but improved the ease and speed of UWF imaging. CONCLUSION: UWF imaging does not increase physiological instability compared to BIO in non-ventilated infants. Specialised swaddling aids may facilitate the imaging procedure. IMPACT: ROP screening can be distressing for premature infants and induce physiological instability during and after the examination. We deployed non-contact ultra-widefield retinal imaging as the default method of ROP screening and show that it induces comparable physiological responses as traditional indirect ophthalmoscopy in non-ventilated babies. Dandle WRAP swaddling facilitated handling and speed of retinal imaging. The study demonstrates that imaging-based ROP screening is safe and efficacious in non-ventilated neonates, and continuous multimodal physiological recordings can provide detailed assessment of the effects of procedures and medications.