In this paper, we investigate the feasibility of high-frequency photoacoustic (PA) imaging to study the shear rate dependent relationship between red blood cell (RBC) aggregation and oxygen saturation (SO2) in a simulated blood flow system. The PA signal amplitude increased during the formation of aggregates and cyclically varied at intervals corresponding to the beat rate (30, 60, 120, 180 and 240 bpm) for all optical wavelengths of illumination (750 and 850 nm).The SO2 also cyclically varied in phase with the PA signal amplitude for all beat rates. In addition, the mean blood flow velocity cyclically varied at the same interval of beat rate, and the shear rate (i.e. the radial gradient of flow velocity) also cyclically varied. On the other hand, the phase of the cyclic variation in the shear rate was reversed compared to that in the PA signal amplitude. This study indicates that RBC aggregation induced by periodic changes in the shear rate can be correlated with the SO2 under pulsatile blood flow. Furthermore, PA imaging of flowing blood may be capable of providing a new biomarker for the clinical application in terms of monitoring blood viscosity, oxygen delivery and their correlation.