The fluidic force microscope (FluidFM) can be considered as the nanofluidic extension of the atomic force microscope (AFM). This novel instrument facilitates the experimental procedure and data acquisition of force spectroscopy (FS) and is also used for the determination of single-cell adhesion forces (SCFS) and elasticity. FluidFM uses special probes with an integrated nanochannel inside the cantilevers supported by parallel rows of pillars. However, little is known about how the properties of these hollow cantilevers affect the most important parameters which directly scale the obtained spectroscopic data: the inverse optical lever sensitivity (InvOLS) and the spring constant (k). The precise determination of these parameters during calibration is essential in order to gain reliable, comparable and consistent results with SCFS. Demonstrated by our literature survey, the standard error of previously published SCFS results obtained with FluidFM ranges from 11.8% to 50%. The question arises whether this can be accounted for biological diversity or may be the consequence of improper calibration. Thus the aim of our work was to investigate the calibration accuracy of these parameters and their dependence on: (1) the aperture size (2, 4 and 8 µm) of the hollow micropipette type cantilever; (2) the position of the laser spot on the back of the cantilever; (3) the substrate used for calibration (silicon or polystyrene). It was found that both the obtained InvOLS and spring constant values depend significantly on the position of the laser spot. Apart from the theoretically expectable monotonous increase in InvOLS (from the tip to the base of the cantilever, as functions of the laser spot's position), we discerned a well-defined and reproducible fluctuation, which can be as high as ±30%, regardless of the used aperture size or substrate. The calibration of spring constant also showed an error in the range of -13/+20%, measured at the first 40 µm of the cantilever. Based on our results a calibration strategy is proposed and the optimal laser position which yields the most reliable spring constant values was determined and found to be on the first pair of pillars. Our proposed method helps in reducing the error introduced via improper calibration and thus increases the reliability of subsequent cell adhesion force or elasticity measurements with FluidFM.