Introduction: An efficient removal of biofilm plays a major role in the prevention of caries as well as gingivitis and periodontitis. In this respect, besides the professional hygiene measures performed in the dental practice, an effective, self-responsible, home-based oral hygiene should also take place. A predilection site for caries and gingivitis is represented by the tooth surfaces below the proximal contact points, which can hardly be cleaned with toothbrushes alone. The aim of the present cross-over pilot study was to investigate whether two-times brushing in accordance with the CIOTIPlus-System (Chewing, Inside, Outside, Tongue and Interdental area, Plus: second brushing) using different brushing regimens (time and type of interdental hygiene tools) has an effect on interdental cleaning (IDC). Methods: 15 subjects (7 females, 8 males, mean age 50.1 ± 6.5 years) were included in this study with a split-mouth design. On five appointments, each of which was preceded by a 72-hour plaque accumulation phase, ten brushing regimens were evaluated on their ability to clean the approximal surfaces; these regimens employed the use of a standard manual toothbrush and interdental hygiene tools. Six brushing regimens using the sequence "brushing – IDC- brushing" (flossing vs. flossing interdental brushing vs. interdental brushing vs. soft picks vs. interdental brushing from vestibular and oral with or without gel) and four brushing regimens using the sequence "IDC – brushing – brushing" (flossing vs. interdental brushing vs. flossing interdental brushing vs. soft picks) were tested. The participants were instructed to brush their teeth according to the "CIOTIPlus"-System. The Quigley-Hein Index (QHI) and the modified Approximal Plaque Index (QH-API) were determined at three time points in order to assess plaque reduction: before brushing (t0), after the first brushing and IDC (t1) as well as after the second brushing (t2). Results: At t1, a significant reduction of the QHI and QH-API values was observed in all groups compared to t0. The highest reduction of the QH-API was observed in the group "brushing – interdental brushing from vestibular and oral – brushing" (BI2B) (ΔQH-API-t0-t1: 2.44 ± 0.45). At t2, the QHI and QH-API values were further significantly reduced in all groups. The greatest reduction of the QH-API was once again observed in the group BI2B (ΔQH-API-t0-t2: 3.16 ± 0.41). However, after the second brushing, the group differences were very small (except for BI2B). Conclusion: Interdental brushing from vestibular and oral seems to be the most effective IDC regimen for reducing the approximal plaque values. Second brushing, as part of the "CIOTIPlus"-System, leads to a higher plaque reduction on smooth and approximal surfaces compared to the one-time brushing, no matter what kind of interdental hygiene tools/ brushing regimens are used. Therefore, any cleaning performance deficits associated with the use of different hygiene tools in the approximal surfaces could be compensated using this approach. Keywords: CIOTIPlus-System, interdental cleaning, plaque reduction, second brushing