This study investigated the effect of the period of use and location of intraoral appliances on enamel surface loss. This randomized, single blind in situ study was conducted in 2 crossover phases based on the period of use, in which maxillary and mandibular appliances were simultaneously worn. Bovine enamel blocks (n = 120) were randomly divided among the studied groups by surface hardness. In each phase, fifteen volunteers used one maxillary appliance and two mandibular appliances for 5 days. Erosive challenge was performed 4X/day by immersion in 0.01 M HCL for 2 minutes. In the continuous phase, the intraoral appliances were worn for 20 hours. In the intermittent phase the appliances were worn for 8 hours and 30 minutes. Enamel loss was determined profilometrically. The discomfort of use of the appliances were evaluated in a questionnaire. Data were analyzed by two-way ANOVA/Tukey's test and chi-square test (p<0.05). The maxillary appliance promoted higher enamel loss compared to the mandibular one (p<0.001). Intermittent use of appliances resulted in similar enamel loss to the continuous one (p = 0.686). All volunteers preferred to use the maxillary appliance in an intermittent regimen. The intermittent use of maxillary appliance is a simplified reliable protocol appropriated for in situ erosion studies in enamel.