The purpose of this study was to perform in vivo animal tests of a novel ventilator prototype for Total Liquid Ventilation (TLV). A total of 17 preterm lambs (gestational age 110±5 days; body weight 3.1±1.2kg), under general anesthesia, were instrumented to record hemodynamic values and gas exchange which were monitored throughout the experiments. Tracheostomy and intratracheal intubation were performed, as well as instrumentation with ex uterus intrapartum treatment (EXIT) support. All fetuses were maintained connected to the placental circulation for the first 10 minutes of ventilation. After complete extraction and disconnection from the mother circulation, the lambs were ventilated for 6 hours. Eleven lambs were ventilated with the prototype ventilator (TLV group); the other 6 lambs (gas ventilation, GV group) were ventilated, as control, with a conventional mechanical ventilator (Siemens Servoventilator 900B). The TLV prototype consists of a volume-controlled, pressure-limited ventilator; it comprises a double-piston system performing respiratory cycles and an uncoupled refresh circuit to achieve perfluorocarbon (PFC) filtration, thermo-regulation, oxygenation, and CO2washout. TLV was performed using PFC FC77 (3M Corp.). At the end of each experiment, fetal lungs were excised and processed for light microscopy to verify airway and lung parenchyma condition. Adequate oxygenation was achieved, while hypercapnia was shown in both groups. Mean arterial pO2 and pCO2 were respectively 72.6 mmHg and 77.8 mmHg, in the TLV group, while 86.6 mmHg and 67.9 mmHg, in the GV group. Higher mortality occurred in the gas ventilation group (67%) than in the TLV group (45%). The histological analyses highlighted a more homogeneous distribution of the respiratory medium in the TLV-ventilated animals (49.3%) with respect to the control group (38.8%) and the histological analyses showed minimal signs of inflammation of the airways and lung parenchyma in the TLV-ventilated animals. The outcomes of the experiments showed the capacity of the TLV ventilator to support the life of very preterm neonatal animals, on the other hand it could be useful to refine the approach to the firsts breathes to minimize the possible lungs damages and improve distribution of the respiratory medium.