To identify early markers of necrotizing enterocolitis (NEC), we hypothesized that continuous abdominal near-infrared spectroscopy (A-NIRS) measurement of splanchnic tissue oxygen saturation and intermittent plasma intestinal fatty-acid binding protein (pI-FABP) measured every 6 hours can detect NEC prior to onset of clinical symptoms. Premature piglets received parenteral nutrition for 48-hours after delivery, followed by enteral feeds every three hours until death or euthanasia at 96-hours. Continuous A-NIRS, systemic oxygen saturation (SpO2), and heart rate were measured while monitoring for clinical signs of NEC. Blood samples obtained at 6-hour intervals were used to determine pI-FABP levels by ELISA. Piglets were classified as fulminant-NEC (f-NEC), non-fulminant-NEC (nf-NEC) and No-NEC according to severity of clinical and histologic features. Of 38 piglets, 37% (n=14) developed nf-NEC, 18% (n=7) developed f-NEC and 45% (n=17) had No-NEC. There were significant differences in baseline heart rate (p=0.008), SpO2 (p<0.001) and A-NIRS (p<0.001) among the three groups. A-NIRS values of NEC piglets remained lower throughout the study with mean for f-NEC of 69±3.8%, 71.9±4.04% for nf-NEC, and 78.4±1.8% for No-NEC piglets (p<0.001). A-NIRS <75% predicted NEC with 97% sensitivity and 97% specificity. NEC piglets demonstrated greater variability from baseline in A-NIRS than healthy piglets (10.1% vs. 6.3%; p=0.04). Mean pI-FABP levels were higher in animals that developed NEC compared to No-NEC piglets (0.66 vs. 0.09 ng/mL;p<0.001). In f-NEC piglets, pI-FABP increased precipitously after feeds (0.04 to 1.87 ng/mL;p<0.001). pI-FABP levels increased in parallel with disease progression and a value >0.25ng/mL identified animals with NEC (68% sensitivity and 90% specificity). NIRS is a real-time, non-invasive tool that can serve as a diagnostic modality for NEC. In premature piglets, low A-NIRS in the early neonatal period and increased variability during initial feeds are highly predictive of NEC, which is then confirmed by rising plasma I-FABP levels. These modalities may help identify neonates with NEC prior to clinical manifestations of disease.