This preliminary in vitro study evaluated the simulated retention force of O-rings used for mini-implant overdenture treatment and the effect of mechanical fatigue corresponding to 6 months of wear. A mandibular overdenture analog device was attached to two mini-implants and underwent a tensile strength test before and after the application of insertion-removal and simulated masticatory cycles. Insertion-removal cycles led to a retention loss of 24%, whereas masticatory cycles did not influence retention. Micro- CT scans showed minor deformation following mechanical cycling, but the changes were milder than those observed in similar clinical specimens. Both experimental loading methods led to physical changes in the O-rings, which explains in part the similar clinically observed wear.