A pulsatile mock loop system was designed and tested. This prototype represents a versatile, adjustable, and controllable experimental apparatus for in vitro studies of devices meant to interface with the human circulatory system. The pumping system consisted of a ventricular chamber featuring two biomorphic silicone valves as the inlet and outlet valves. The chamber volume is forced by a piston pump moved by a computer-controlled, low-inertia motor. Fluid dynamic tests with the device were performed to simulate physiological conditions in terms of cardiac output (mean flow of 5 and 6 L/min, with beat rates from 60 to 80 bpm), of rheological properties of the processed fluid, and of systemic circulation impedance. The pulsating actuator performed a good replication of the physiological ventricular behavior and was able to guarantee easy control of the waveform parameters. Experimental pressure and flow tracings reliably simulated the physiological profiles, and no hemolytic subatmospheric pressures were revealed. The performance of the prototype valves was also studied in terms of dynamic and static backflow, effective orifice area, and pressure loss, resulting in their applicability for this device. Mechanical reliability was also tested over 8 h. The device proved to be a reliable lab apparatus for in vitro tests; the pumping system also represents a first step toward a possible future application of pulsating perfusion in the clinic arena, such as in short-term cardiac assist and pulsatile cardiopulmonary bypass.