Limited information is currently available relative to the effect of masticatory loads on the retentive properties of Locator attachments. The aims of this in vitro study were to assess and compare the effect of simulated mastication on the retention of white, pink, and blue Locator inserts for overdentures retained by 2 implants. Thirty specimens simulating a nonanatomic edentulous flat ridge with two implants and an overdenture were divided into 3 groups according to the color of the fitted insert: transparent clear group (n = 10), pink group (n = 10), and blue group (n = 10). Retention forces were measured in an axial direction initially and after 100,000 cycles of simulated masticatory loads. One-way ANOVA followed by Tukey's post hoc tests were used to compare retention values and percentage retention loss between the 3 groups with significance set at p = 0.05. The 3 groups presented significant differences in retention at baseline (9.95 ± 1.91 N, 15.43 ± 4.08 N, and 41.73 ± 9.29 N for the blue, pink, and clear groups, respectively) and after simulated mastication (6.37 ± 2.64 N, 14.00 ± 3.89 N, 38.20 ± 5.11 N for the blue, pink, and clear groups, respectively). Within the same group, cyclic loading did not significantly affect retention in the clear and pink groups, while the blue inserts showed a significant retention loss (-37%) after loading. The results suggest that short-term simulated mastication affects the extra-light blue inserts but not the more-retentive inserts. © 2017 by the American College of Prosthodontists.