Introduction: The cleaning efficacy of different toothbrushes is often tested in a plaque regrowth crossover design, i.e. participants have to abstain from oral hygiene, repeatedly. This might influence the amount of plaque accumulation. As the baseline plaque is the most important confounder for the amount of plaque reduction those changes would significantly influence the outcome of the study. Aim of the study: The aim of the study was to investigate whether or not a crossover design would influence the 24-hours plaque regrowth. Materials and Methods: In two separate toothbrush studies with a crossover design, 158 partici-pants volunteered to abstain twice (T1 and T2) for 24 hours from all oral hygiene pro-cedures prior to the test. The time between the two arms of each study was 12 days. Plaque accumulation was measured by one calibrated examiner using the Turesky modification of the Quigley & Hein Index. Results: The overall 24-hours plaque regrowth resulted in scores of 2.10± 0.60 (T1), and 1.67± 0.56 (T2; p Keywords: plaque regrowth, crossover design, toothbrush study