Blood biophysical properties including blood viscosity and RBC (red blood cell) aggregation have been considered as promising bio-markers for effectively monitoring cardiovascular diseases. In this study, simultaneous measurement of blood viscosity and RBC aggregation is suggested under in-vitro circulation loop. By operating pinch valve as open and close (Topen=100 s, Tclose=300 s) for each period, blood is supplied from in-vitro circulation to a microfluidic device periodically. When pinch valve is released, blood viscosity is quantified by monitoring interface between blood (Normal RBCs in plasma, hematocrit=30%) and PBS in single microfluidic channel. Then, RBC aggregation is calculated by using temporal variations of image intensity within a specific ROI, with the pinch valve closed. As a result, blood viscosity and RBC aggregation are remained constant over time. Thus, it is found that the suggested method can be employed to evaluate temporal variations of biophysical properties of blood circulated under in-vitro closed loop.