BACKGROUND: Sepsis remains the largest preventable source of neonatal mortality in the world. Heart rate variability (HRV) analysis and noninvasive cardiac output have been shown to be useful adjuncts to sepsis detection in many patient groups. METHODS: With Institutional Review Board approval, 4 septic and 6 nonseptic extremely low birth weight patients were enrolled. Data from septic and healthy patients were collected for 5 hours. Electrocardiogram waveform and traditional vital signs were collected and the RR intervals were calculated; then HRV analysis was performed in both the time and frequency domain. RESULTS: HRV measurements in time domain, heart rate, and pulse oximetry (SpO2) were significantly different in septic patients vs nonseptic controls. CONCLUSIONS: These results indicate that nonconventional vital signs such as HRV are more sensitive than traditionally used vital signs, such as cardiac output and mean arterial pressure, in the confirmation of sepsis in extremely low birth weight neonates. HRV may allow for earlier identification of septic physiology.