A bidirectional optical (BDO) three-stage prism grating system has been developed to measure online direction and magnitude of red blood cell (RBC) velocities in the microcirculation. The paper describes a mathematical evaluation of the BDO system. The optical grating configuration was simulated in terms of a spatial filter. The input signal was the simulated spatial light distribution of a group of moving RBCs Three methods for estimating the mean frequency (fmean) of the signal corresponding to RBC velocity were evaluated on the criteria of accuracy and noise sensitivity. All underestimated the RBC velocity. The reference method calculates fmean from the power density spectrum. A zero-crossing counting technique and a method based on time differentiation underestimated the mean frequency by 14·1±11·3 per cent (mean ±SD) and 0·9±0·1 per cent, respectively. The zero-crossing counting technique was more sensitive to noise than both other methods. The reference method, operating in the frequency domain, is preferred, because the power density spectrum obtained can be corrected for filtering errors induced by higher-order passbands of the optical filter.