The differences in the roller diameters within a roller bearing are unavoidable in practice, which is known as the off-sized effect. The off-sized effect results in the dynamic variation of the load distribution in bearings and further the abnormal vibration and premature failure of bearings. This paper experimentally investigates the effect of off-sized rollers on the dynamic load distribution in a cylindrical roller bearing. Different configurations of off-sized rollers are given considering the oversized and undersized roller conditions, and the number and position of off-sized rollers. The real-time roller-raceway contact loads distributed in the bearing are measured under these test cases. The results show that a single off-sized roller leads to the loading change directly on itself and further the load rebalances among the adjacent rollers. Under the conditions of multiple off-sized rollers, a stack effect of the competition rule of roller loading dominates the variation range of the dynamic load distribution. As results of the stack effect, among all roller configurations, the configuration of multiple off-sized rollers adjacent to each other leads to the minimum range of the dynamic load distribution, while the configuration of two off-sized rollers separated by a regular roller leads to the maximum one.