To evaluate the effect of two molar clamps on artificially demineralized enamel of permanent teeth after three periods of isolation.Twenty extracted third molars were immersed in a biological solution for development of artificial white spot lesions and then were equally divided into two groups according to the clamp being placed (Group G205: clamp no. 205; Group G26: clamp no. 26). The loss of fluorescence and lesion area of demineralized surface after each isolation were assessed by quantitative light-induced fluorescence.There were significant statistical differences in artificially induced white spot lesions after the second and third clamp placement with both clamps. There was also a significant difference in quantitative light-induced fluorescence readings between groups when molar clamps were placed for the third time.It is possible to lose dental structure when using rubber dam clamps on teeth with white spot lesions in the cervical enamel.