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The spatial irregularities of red blood cells (RBCs) in continuous pulsing flow condition in micro-channels were investigated by analyzing the time variability of optical signatures obtained by recording transmitted light variability at specific location in micro-flow channels. Different flow conditions were filmed and analyzed by the digital particle image velocimetry (DPIV) to characterize local flow velocity across the whole micro-channel and in four sub-areas selected to study the particles behaviors close to the walls and in the micro-channel bulk. Starting from a behavioral classification based on the three flow patterns identified as { Weak Activity, Vorticity, Alignment }, an analysis to detect the spatial irregularity in the flow distribution was carried out. The velocity gradients and four nonlinear parameters (shear rate, strain rate, vorticity, divergence) were computed from the time-varying velocity maps obtained by DPIV and used to provide a quantitative characterization of the flow features. The comparison of the results obtained in the four experiments has made possible an overall understanding of the RBC movements in different conditions and, as well, the establishment of a analysis procedure for flows spatial irregularity detection.