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Purpose: To evaluate the marginal integrity of various pit and fissure sealants subjected to different application methods. Methods: A total of 253 non-carious human third molars extracted and randomly divided into two groups according to the preparation method employed: invasive or non-invasive. Eight fissure sealant materials were tested: nano-filled flowable composite (Filtek Ultimate Flow), nanohybrid flowable composite (GrandioSo Flow), micro-hybrid flowable composite (Majesty Flow), resin-based unfilled fissure sealant (ClinPro Sealant), resin-based filled fissure sealant (Fissurit FX), resin-based highly filled fissure sealant (GrandioSeal), giomer-based fissure sealant (BeautiSealant), and glass ionomer-based fissure sealant (Fuji Triage). Samples were subjected to two-year cyclic thermo-mechanical and brushing simulations. Two observers quantitatively evaluated the restoration margins and classified them as either "permanent restoration edge", or if a gap larger than 250 μm was evident, "gapping at the restoration edge". The extent of the gap was recorded as a percentage relative to the total length of the restoration edge. Results:The baseline marginal adaptation had no significant effect on the marginal adaptation (P > 0.05). However, the preparation method and type of fissure sealant material had a significant impact on the marginal adaptation (P < 0.05). Conclusion:On the basis of quantitative analysis, the highest marginal integrity was observed for flowable composites, whereas the lowest was observed for glass ionomer-based fissure sealant.