Objective Identify clinical conditions associated with a large increase (spike) in the heart rate characteristics index in VLBW infants. Study design Retrospective medical record review within a day of all large heart rate characteristics index spikes (increase of ≥3 from prior 5 day average) in VLBW infants at a single center enrolled from 2007–2010 in a multicenter trial of heart rate characteristics monitoring. In the trial, infants were randomized to having their heart rate characteristics index displayed to clinicians or not displayed. Results Of 274 eligible infants, 224 large heart rate characteristics spikes occurred in 105 infants. Thirty-three spikes were associated with surgery or procedures requiring anesthetic or anticholinergic medications, and infection-related conditions were the most common clinical association with the other spikes. Of the first spikes in 47 infants randomized to conventional monitoring (heart rate characteristics index not displayed to clinicians), 53% were associated with suspected or proven infection. Respiratory deterioration without suspected infection occurred with 34%, and no association was identified in 13%. Infants randomized to having their heart rate characteristics index displayed were more likely to have antibiotics initiated around the time of a large heart rate characteristics index spike. Conclusions Sepsis, other infectious or systemic inflammatory conditions, respiratory deterioration, and surgical procedures are the most common clinical associations with a large increase in the heart rate characteristics index in VLBW infants. This information may improve use of heart rate characteristics monitors in NICU patients.