By using a systems toxicology approach, the effect of repeated cigarette smoke (CS) exposure was investigated in four different human organotypic models mimicking in vitro the bronchial, nasal, buccal and gingival epithelium which are in direct contact with CS upon inhalation in vivo. Such assay systems are relevant models to assess the disease-risk reduction potential of novel modified risk tobacco products. In this study, the smoking behaviour of a light smoker during one day was recapitulated by exposing repeatedly and in parallel four human tissue culture models (bronchial, nasal, buccal and gingival epithelium) directly at the air/liquid interface (VITROCELL® system) to four cigarettes with one hour intervals between each cigarette. These organotypic cultures are produced from primary fibroblasts and epithelial cells derived from non-smoking donors and reproduce as closely as possible the in vivo tissues characteristics (e.g., morphology, cell types composition). All tissues were first simultaneously exposed to various CS concentrations from 8% to 35% (diluted vol/vol with humidified air) or to humidified air (control). Gingival tissues were more sensitive to CS exposure than the other tissues based on a cell viability assay. Then, different endpoints (e.g., gene and microRNA expression, CYP activity, pro-inflammatory markers release, differential cell counts, cytotoxicity measurement) were captured at various post-exposure times from each tissues exposed in a second run to only two doses of whole CS (10% and 16% - doses inducing less than 20% cell death) or to humidified air. The impact of repeated exposure of CS was finally analyzed in each tissue culture using an integrative systems biology approach allowing to identify, compute and compare the biological perturbations occurring in each in vitro model.