BACKGROUND: Retinopathy of prematurity (ROP) ophthalmologic examinations cause stress and pain. Infants\' stress and pain can be measured non-invasively using skin conductance (SC) and high frequency heart rate variability (HF-HRV), reflecting sympathetic-mediated sweating and parasympathetic activity, respectively. OBJECTIVES: To test the utility of SC to detect sympathetic activation during ROP examination, and the contribution of HF-HRV to predict stability post-examination. METHODS: In this prospective, single center study, we measured SC continuously pre-, during, and post-examination, and HRV at 24 h pre-ROP examination. Clinical data included stability [apneas, bradycardias, and desaturations (A/B/Ds)], and interventions post-examination. RESULTS: SC increased 56% above baseline during ROP examination (p = 0.001) and remained elevated post-examination (p = 0.02). Post-hoc analysis showed higher illness acuity, represented by need for respiratory support, was associated with lower HF-HRV at 24 h pre-ROP examination (p = 0.001). Linear regression indicated lower HF-HRV at 24 h pre-examination contributed to the need for higher intervention (i.e., stimulation to breathe, oxygen support) particularly among infants with higher illness acuity [F(1, 15) = 5.05, p = 0.04; beta = -1.33, p = 0.04]. CONCLUSION: ROP examination induced a 2-fold increase in sympathetic activation which remained above baseline in recovery. Also, we propose that the low parasympathetic tone associated with autonomic imbalance contributes to instability and need for higher intervention to assure stabilization with A/B/D events. Our findings provide insight into the underestimation of adverse events associated with ROP examination and identification of infants who may be more vulnerable to potential sequelae following ROP examinations.