Background: Midazolam sedation and morphine analgesia are commonly used in ventilated premature infants. Objectives: To evaluate the effects of midazolam versus morphine infusion on cerebral oxygenation and hemodynamics in ventilated premature infants. Methods: 11 patients (GA 26.6–33.0 weeks, BW 780–2,335 g) were sedated with midazolam (loading dose 0.2 mg/kg, maintenance 0.2 mg/kg/h) and 10 patients (GA 26.4–33.3 weeks, BW 842–1,955 g) were sedated with morphine (loading dose 0.05 mg/kg, maintenance 0.01 mg/kg/h). Changes in oxyhemoglobin (ΔcO2Hb) and deoxyhemoglobin (ΔcHHb) were assessed using near infrared spectrophotometry. Changes in cHbD (= ΔcO2Hb – ΔcHHb) reflect changes in cerebral blood oxygenation and changes in concentration of total hemoglobin (ΔctHb = ΔcO2Hb + ΔcHHb) represent changes in cerebral blood volume (ΔCBV). Changes in cerebral blood flow velocity (ΔCBFV) were intermittently measured using Doppler ultrasound. Heart rate (HR), mean arterial blood pressure (MABP), arterial oxygen saturation (saO2) and transcutaneous measured pO2 (tcpO2) and pCO2 (tcpCO2) were continuously registered. Statistical analyses were carried out using linear mixed models to account for the longitudinal character study design. Results: Within 15 min after the loading dose of midazolam, a decrease in saO2, tcpO2 and cHbD was observed in 5/11 infants. In addition, a fall in MABP and CBFV was observed 15 min after midazolam administration. Immediately after morphine infusion a decrease in saO2, tcpO2 and cHbD was observed in 6/10 infants. Furthermore, morphine infusion resulted in a persistent increase in CBV. Conclusions: Administration of midazolam and morphine in ventilated premature infants causes significant changes in cerebral oxygenation and hemodynamics, which might be harmful.