Introduction: Despite therapeutic hypothermia (TH), neonates with neonatal encephalopathy (NE) often present with multi-organ dysfunction, including cardiac alterations in the first days of life.Objectives: Describe the cardiac performance of neonates with moderate to severe NE treated with TH using three-dimensional echocardiography (3D-ECHO) relative to a control population without NE.Methods: Single-centre prospective case-control study of neonates with moderate to severe NE treated with TH between 2019 and 2021, compared to healthy neonates. The control group were newborns recruited in the nursery without need for resuscitation. 3D-ECHO was performed on day of life (DOL) 2. Left ventricular (LV) metrics were measured using 3D-Speckle Tracking Echocardiography. Descriptive statistics were used to compare the two groups.Results: We included 16 neonates with NE and 50 controls. Birthweight and gestational age at birth were similar (Table 1). Neonates with NE had lower systolic blood pressure (BP) but similar diastolic BP at ECHO, while 11/16 were exposed to cardiovascular medications at ECHO. These neonates had lower LV end-diastolic volume (4.2 [1.2] vs 5.0 [1.4] mL; p=0.04), lower stroke volume (2.2 [0.7] vs 2.9 [0.8] mL; p=0.002) and lower ejection fraction (52 [6] vs 56 [8] %; p=0.0008). These infants also had lower peak global circumferential strain (-25 [5] vs -28 [5] %; p=0.03). The association between NE and LV-ejection fraction remained significant after adjusting for weight (b=-5.7%, 95% Confidence Interval [-9.2; -2.2], p=0.002).Conclusions: Neonates with moderate to severe NE had reduced LV functional markers on DOL 2 as comparted to healthy controls. They had decreased systolic BP and LV end diastolic volume. These findings may be secondary to the asphyxia, catecholamines and medications exposure, or to TH. Future studies should consider 3D-ECHO as a modality to investigate these patients and the impact of therapeutic interventions.