Background: Many agents and toothpaste formulations have been proposed for treatment of dentine hypersensitivity. However, studies have reported equivocal or contradictory results, potentially related to a lack of clinical efficacy for agents being tested, poor study design or the use of assessment techniques which are unable to distinguish longitudinal changes in dentine hypersensitivity. Aim: To investigate the reproducibility of commonly used measurement protocols for dentine hypersensitivity on single or multiple teeth. In addition, newly-developed controlled air and cold fluid stimuli were investigated. Methods and Materials: Standardised pain stimulation techniques were used in 2 investigations with 63 and 42 subjects, respectively. The reproducibility of perceived hypersensitivity before and following stimulation was recorded over time periods of 14 and 2 days, respectively, using visual analogue scales and threshold techniques. Results: Subject demographics resembled those reported in other hypersensitivity investigations. Analysis using several measures of reproducibility indicated that subject-based reproducibility was limited, even when the stimuli were standardised. However, the data are consistent with the findings of investigations on pain assessment in other fields. Conclusions: The reproducibility of subjects in clinical trials of dentine hypersensitivity may therefore contribute to difficulties in establishing treatment efficacy of agents in clinical trials.