This in situ study aimed to evaluate effects of waiting periods after erosive challenge before toothbrushing on enamel abrasion and nanoindentation hardness. Ten subjects wore intraoral appliances each with a set of 4 bovine enamel blocks. The enamel blocks were subjected to 2 cycles a day for 3 days as follows; intraoral exposure to form acquired pellicle and extraoral erosion followed by either 0, 3, 30 or 60 min intraoral exposure and then brushing, which was performed using an automatic brushing machine. Abrasive loss was assessed by white light interferometry. Nanoindentation was performed to calculate relative hardness. Abrasion and relative hardness were statistically analyzed by ANOVA. Abrasive loss was significantly less in groups exposed to saliva compared with 0 min (p<0.05); there was no significant difference between 30 and 60 min (p>0.05). Relative hardness was statistically higher after intraoral exposure, but no differences existed among any intraoral exposure periods (p>0.05).