Background: Prehospital triage of patients with trauma is routinely challenging, but more so in mass casualty situations and military operations. The purpose of this study was to prospectively test whether heart rate variability (HRV) could be used as a triage tool during helicopter transport of civilian patients with trauma. Methods: After institutional review board approval and waiver of informed consent, 75 patients with trauma requiring prehospital helicopter transport to our level I center (from December 2007 to November 2008) were prospectively instrumented with a 2-Channel SEER Light recorder (GE Healthcare, Milwaukee, WI). HRV was analyzed with a Mars Holter monitor system and proprietary software. SDNN (standard deviation [SD] of the normal-to-normal R-R interval), as an index of HRV, was correlated with prehospital trauma triage criteria, base deficit, seriousness of injury, operative interventions, outcome, and other data extracted from the patients' medical records. There were no interventions or medical decisions based on HRV. Data were excluded only if there was measurement artifact or technical problems with the recordings. Results: The demographics were mean age 47 years, 63% men, 88% blunt, 25% traumatic brain injury, 9% mortality. Prehospital SDNN predicted patients with base excess ≤-6, those defined as seriously injured and benefiting from trauma center care, as well as patients requiring a life-saving procedure in the operating room. No other available data, including prehospital en-route vital signs, predicted any of these. The sensitivity, specificity, positive predictive value, and negative predictive value were 80%, 75%, 33%, 96%, respectively, with and an overall accuracy of 76% for predicting a life-saving intervention in the operating room. Conclusions: This is the first demonstration that prehospital HRV (specifically SDNN) predicts base excess and operating room life-saving opportunities. HRV triages and discriminates severely injured patients better than routine trauma criteria or en-route prehospital vital signs. HRV may be a useful civilian or military triage tool to avoid unnecessary helicopter evacuation for minimally injured patients. A prospective, randomized trial in a larger patient population is indicated.