We assessed reference values in a group of apparently healthy blood donors. A total of 1980 blood donors was recruited and tested for the presence of NT-proBNP using a newly developed electrochemiluminescence immunoassay (ECLIA) method. NT-proBNP clustered in all blood donors below the age of 50 years and an upper limit of normal (ULN) was found to be 84 pg/ml for males and 146 pg/ml for females. Mean NT-proBNP values increased with increasing age which was due to an increasing number of individuals exceeding the ULN. Age- and gender-appropriate NT-proBNP levels decreased with increasing hemoglobin levels. Hemoglobin but not creatinine levels influenced the NT-proBNP concentration in this cohort. The upper limit of normal can be used in clinical studies to further assess groups of diseased individuals to define clinical cutoffs.