This in vitro study evaluated the effect of toothpastes with different active ingredients on dentin permeability using an extended protocol including multiple applications and several thermal ageing cycles in the presence or absence of human saliva. The Null hypothesis was that dentin permeability of a hydroxyapatite containing toothpaste (BR), a potassium nitrate (SP) and an arginine and calcium carbonate (EH) containing toothpaste were similar. Dentin permeability was measured as hydraulic conductance using a commercially available capillary flow system (Flodec, Geneva) and results were expressed as % relative to matching controls. Without saliva, the ranking (best first) of dentin permeability was BR(61%) < SP(87%) < EH(118%), with saliva EH(63%) < SP(72%) < BR(88%). Saliva increased or decreased permeability dependent upon the test material. BR reduced dentin permeability significantly more in absence of saliva, with saliva EH was superior to BR. Repeated material application decreased and thermal ageing increased dentin permeability. The different tooth pastes reduced permeability differently, the best being BR without saliva, the least EH without saliva. The newly introduced test conditions (ageing, saliva, multiple applications) influenced single results significantly, and as they better simulate the in vivo situation they should be considered to be included in further in vitro permeability testing of desensitizing preparations.