The clinical relevance of brain natriuretic peptide (BNP) and N-terminal (NT)-proBNP assays as a diagnostic tool and prognostic marker in patients with cardiovascular diseases has recently been confirmed. However, several studies demonstrated variation of intra-individual BNP concentrations of >30% (ranging from 30% to 50%) with reference change values at the 95% confidence interval (i.e., the estimated critical difference) ranging from 99% to 130% in healthy subjects and heart failure patients. According to this estimated confidence interval, only a great variation in plasma BNP levels should be considered significant in an individual patient (for example, a decrease of >50% or an increase of more than two-fold). Many recent clinical studies have demonstrated that BNP variations below this estimated critical difference could also have clinical relevance. Like the concentration of other neuro-hormones, levels of plasma BNP fluctuate widely and rapidly along with heart rhythm and blood pressure variations in response to physiological stimuli. However, biological variation of BNP should not be interpreted strictly as random fluctuation around a homeostatic set point, as assumed by the common model used in all studies on biological variation of BNP reported in the literature. These results cannot be directly transferred to clinical practice. While awaiting more accurate studies, we suggest that variations of plasma BNP three-fold greater than the analytical imprecision should be considered as potentially relevant from a physiological and clinical point of view.