Currently, substantial variation in epidemiologic studies exists regarding the number of blood pressure (BP) readings obtained and the way in which they are combined. This might result in systematically different BP estimates. We therefore analysed data from 25,891 subjects (10,124 men and 15,767 women) of the EPIC-Potsdam Study (European Prospective Investigation into Cancer and Nutrition) to estimate the magnitude of differences between consecutive BP readings and their combinations. Three measurements with 2 min intervals were performed in the sitting position on the right arm with the supported arm elevated at heart level by trained interviewers using oscillometric devices. Mean BP declined from first to second reading and further to third reading by systolic 5.0/0.9 mmHg in men and 4.9/0.8 mmHg in women and by diastolic 1.5/0.3 mmHg in men and 1.9/0.5 mmHg in women, as well as pulse pressure (PP) (3.5/0.6 in men, 3.0/0.3 in women) and hypertension prevalence (9.1/1.7%-points). The magnitude of BP decline depended on BP level, age, body mass index (BMI), and BP medication. Combinations including the first reading lead to generally higher estimates than subsequent readings or their combination. Published data on mean BP, PP and hypertension prevalence depend on the number and subsequent handling of BP readings which might introduce bias to the comparison of different studies unless the same defined readings were used. The combination of the second and third reading seems to be favourable over any single reading or other combinations.