Masticatory function can be evaluated objectively as the capacity of an individual to fragment solid food after a fixed number of chewing cycles, the so-called masticatory performance (MP). The objective of this study was to evaluate the reliability of four different test materials (Optosil, Optocal, Zetapuls, and Perfil) and five disinfection protocols by aspersion and immersion (no disinfection, 2% glutaraldehyde, 2% chlorhexidine, 5.25% sodium hypochlorite, and 70% alcohol) on the MP, determined at three moments (24 hours, 15 and 60 days) after storing the fragmented blocks. MP was evaluated by calculating X50 through the sieving technique and the Rosim-Ramler equation. The weight and microbiologic count (colony forming units, CFUs) of chewed blocks were measured to identify any variations that would make MP determination unfeasible. Differences in MP were observed among the materials (p < 0.01). Perfil presented the highest X50 value (worst MP determination), followed by Zetaplus (both p < 0.05), Optosil, and Optocal (both p > 0.05). The time and disinfection type had no influence on MP (p > 0.05). The number of CFUs differed between the nondisinfected group and all other disinfection groups at all time points (p < 0.01). No other significant difference in CFU count between disinfection groups was observed. In conclusion, disinfection did not alter the reliability of the test materials for the MP calculation for up to 60 days.