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A repeated measures digital imaging technique (Digital Plaque Image Analysis) was used to assess variations in plaque formation, including levels of plaque developed following evening and morning tooth brushing with a standard dentifrice, to establish a baseline for future assessments of antimicrobial formulations.Following a rigorous oral hygiene period, subjects were provided with a standard commercial (non-antibacterial) dentifrice and manual toothbrush and instructed to brush b.i.d., as normal. On six separate days over two weeks, subjects reported at three times for a daily plaque assessment: in the morning before oral hygiene, post-brushing, and in the afternoon post-brushing.Morning plaque levels covered approximately 10% of the measured dentition, and plaque was removed by 75% with morning tooth brushing. Plaque underwent rapid regrowth during the day, and averaged approximately 7% coverage by the afternoon.These results support the value of Digital Plaque Image Analysis in recording diurnal plaque variations and treatment effects, and suggest that assessment of oral hygiene efficacy (either mechanical or chemopreventive) accounts for diurnal variations in plaque formation. In addition, the results suggest that plaque regrowth and virulence activity overnight is a significant target for oral hygiene interventions.