BACKGROUND: Necrotizing enterocolitis (NEC) is the leading cause of death due to gastrointestinal disease in preterm neonates; yet, clinicians lack reliable and noninvasive predictive tools. PURPOSE: We aimed to test that diminished high-frequency heart rate variability (HF-HRV) and elevated levels of proinflammatory cytokines would have utility in NEC prediction. METHODS: In this multisite prospective study, we enrolled 250 preterm (26-34 weeks\' postmenstrual age [PMA]) neonates with physiological stability at 72 hours of life. HRV was measured noninvasively using electrocardiograhic data from standardized cardiorespiratory monitors at postnatal week 1 of life and weekly thereafter until 35 weeks\' PMA or discharge; blood was collected for cytokines at postnatal weeks 1 and 3. NEC was diagnosed via Modified Bell\'s Staging Criteria. RESULTS: HF-HRV was decreased at weeks 1 and 2 in neonates (47% females) who developed feeding intolerance or stage 2+ NEC. In addition, these neonates displayed elevated levels of IL-8 at week 1 and increased levels of IL-1beta, IL-6, TNF-alpha, and IL-8 at week 3 of life. Low HF-HRV was associated with elevated IL-6 or IL-8 levels at weeks 1 and 3 of life. Logistic regression indicated that only HF-HRV was a significant predictor of feeding intolerance or NEC development. IMPLICATIONS FOR PRACTICE AND RESEARCH: HRV is a promising noninvasive modality for NEC risk detection. The association of low HF-HRV with elevated proinflammatory cytokines provides evidence for a putative role of the vagal cholinergic pathway in NEC pathogenesis. Future studies should focus on application of these techniques to test clinical therapeutics.Video Abstract available at https://journals.lww.com/advancesinneonatalcare/Pages/videogallery.aspx?autoPlay=false&videoId=54.