BACKGROUND: Studies indicate that fasting and diurnal variation can influence serum Chromogranin A (CgA) concentrations and furthermore, stability of the CgA molecule has been questioned. We investigated the impact of pre-analytical conditions on a CgA assay. METHODS: Serum CgA concentrations were measured in blood samples collected from ten healthy subjects at three time points during three days with and without fasting. Samples were kept at 4°C and re-measured after 24 and 48 h in order to test the stability of the measured epitopes. For establishment of a reference interval, serum samples were collected from 120 healthy individuals. RESULTS: Serum CgA concentrations did not vary between fasting and non-fasting individuals (p=0.74), nor between gender (p=0.66). The analytical imprecision was 4.3%, the within-subject variability was 11.9%, while the between-subject variability was as high as 50.2%. Serum CgA concentrations measured during the day did however not differ (p=0.30). Of importance, re-analysis at 4°C showed significant decay in the CgA concentrations after 24 h (mean decrease 15.6%) and 48 h (mean decrease 44%). The median CgA concentration in 120 healthy subjects was 41.2 ng/mL with a reference interval of 30.3-94.4 ng/mL (2.5-97.5 percentile). CONCLUSIONS: Fasting does not influence CgA concentrations significantly, while sample stability is a major issue with the assay tested. The biological variation of CgA was defined and a reference interval was established.