Purpose: This study investigated the influence of several methodological details on the shear bond strength (SBS) testing of pit and fissure sealants. The following variables were considered: type of enamel surfaces, prismatic vs aprismatic enamel, etching time, and aging and shearing procedures. Materials and Methods: The surfaces of 180 healthy human third molars were divided into 4 different sections (mesial, distal, buccal, and oral). After tooth separation, the specimens were randomly allocated to the following groups: 1) enamel preparation: prismatic vs aprismatic; 2) etching: 30 s vs 60 s; 3) aging: 1 day or 3 months vs 5000 thermocycles; 4) shearing: notched-edge method (ISO 29022:2013) vs knife-edge method. After following each protocol, SBS was determined using a universal testing machine, followed by failure mode analysis. Data were analysed using Mann-Whitney U-tests and regression analyses. Results: In the aprismatic enamel group, the longer etching time resulted in slightly, not statistically significantly higher SBS. When aging sealants on aprismatic enamel with different procedures, significantly lower SBS was found for 5000 thermocycles. In the case of aprismatic enamel etched for 60 s and sheared with the notched-edge blade, there was no significant difference between the aging procedures. Failure mode analysis showed adhesive failures to be predominant. Simple linear regression revealed that all of the included factors significantly influenced SBS. In a multiple linear regression model, the variables "aprismatic enamel" and "distal surface" were associated with a higher SBS; "5000 thermocycles" reduced SBS significantly. Conclusion: Enamel grinding, aging method, and type of enamel surface significantly influenced the SBS. Keywords: pit and fissure sealants, shear bond strength, mechanical testing, methodology