We previously reported that polybutylene terephthalate (PBT) toothbrushes become less effective for plaque removal after two months of use. However, it remains unknown how the bristle stiffness of PBT toothbrushes changes after several months of use. We performed a single-center randomized controlled trial to evaluate the bristle stiffness and bristle splaying of soft and medium manual toothbrushes among dental and medical students of Hiroshima University. Subjects were 80 participants who met the criteria. Participants were randomly assigned to the soft toothbrush group (n = 40) or the medium toothbrush group (n = 40). We collected toothbrushes immediately after first use (T0), after one month of use (T1), after two months of use (T2), and after three months of use (T3). Bristle stiffness was measured according to the International Organization for Standardization (ISO) 22254. The mean bristle stiffness (N/cm2) of soft and medium toothbrushes was significantly lower at T2 and T3 than at T0 (T2 vs. T0, soft; 3.63 vs. 3.89, P < 0.01 and medium; 4.33 vs. 4.52, P < 0.05, respectively, and T3 vs. T0, 3.62 vs. 3.89, p < 0.01 and 4.18 vs. 4.52, p < 0.001, respectively). Bristle stiffness was significantly reduced in soft and medium PBT toothbrushes after two months of use.