OBJECTIVES: The autonomic nervous system (ANS) can both modulate and be modulated by the inflammatory response during critical illness. We aimed to determine whether heart rate variability (HRV), a measure of ANS function, is associated with proinflammatory biomarker levels in critically ill children. DESIGN: Two cohorts were analyzed. The first was a prospective observational cohort from August 2018 to August 2020 who had plasma proinflammatory cytokine measurements within 72 hours of admission, including tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6, and IL-8. The second was a retrospective cohort from June 2012 to August 2020 who had at least one C-reactive protein (CRP) measurement within 72 hours of admission. SETTING: Forty-six-bed PICU. PATIENTS: Critically ill children in either cohort who had continuous heart rate data available from the bedside monitors. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Sixty-two patients were included in the prospective cohort and 599 patients in the retrospective cohort. HRV was measured using the age-adjusted integer heart rate variability (HRVi), which is the sd of the heart rate sampled every 1 second over 5 consecutive minutes. The median HRVi was measured in the 12-hour period ending 30 minutes prior to inflammatory biomarker collection. HRVi was inversely correlated with IL-6, IL-8, and CRP levels (p </= 0.02); correlation with IL-8 and CRP persisted after adjusting for Pediatric Risk of Mortality III and age, and median HR and age (p < 0.001). CONCLUSIONS: HRVi is inversely correlated with IL-6, IL-8, and CRP. Further studies are needed to validate this measure as a proxy for a proinflammatory state.