This in-vitro study aimed to evaluate the retentive force and dislodgment time of three stud attachment systems used for mandibular two-implant overdentures by simulating insertion/removal cycles. From a simulation of a completely edentulous mandible with two parallel implants (Ø4.5 mm internal hex connection) (Zimmer Biomet, Warsaw, IN), 15 resin bases were fabricated and divided into three groups (n = 5 each): OT Equator (Rhein83, Bologna, IT), Locator (Zest Dental Solutions, Escondido, CA) and Locator R-Tx (Zest Dental Solutions, Escondido, CA). Pink inserts underwent 2000 cycles of thermocycling (SD MECHATRONIK GmbH, Feldkirchen-Westerham, Germany) and were soaked in citric acid for 24 days in an incubator. Each base underwent 2000 insertion/removal cycles on the Versa Test testing machine (Mecmesin Ltd., W. Sussex, UK) which was used to measure the retentive force and dislodgment time. The results were analyzed using ANOVA followed by the post-hoc Tukey test, Kruskal-Wallis test, and Pearson correlation coefficient test. A p-value of < 0.05 was considered statistically significant. The retentive force and dislodgment time of the three systems significantly decreased over the cycles (p < 0.05). The Locator R-Tx showed significantly greater retentive force ​​than did the other systems (p < 0.05), except for the last cycles wherein no significant difference was found with the OT Equator (p > 0.05). The OT Equator had the most stable retention over the cycles (p > 0.05; cycle 1500). The three systems showed satisfactory retentive force ​​during the 2000 cycles. The Locator R-Tx demonstrated the best retention, while the OT Equator exhibited the most stable retention over time.