The sweat test remains the gold standard to diagnose CF and it is a crucial outcome used to monitor the response to CFTR-modifying new therapies. But it’s not completely clear the biological variability of chloride in sweat both in patients with CF and in healthy subjects. This information is essential to better evaluate both the efficacy of new therapies and the real diagnostic role of sweat chloride in suspicion of CF. This was a two-center prospective clinical study, aimed to investigate the sweat chloride intra-individual biological variability both in subjects with CF and in healthy people. 36 subjects aged 6 to 18 years were selected, enrolled and divided into 3 groups: 12 patients with CF, 12 patients with CFTR-RD, and 12 healthy subjects. Possible correlation of the test values with different conditions (sex, age, season, diet, menstrual cycle) was studied by means of repeated sweat tests, performed in different conditions. At the end of data collection, a statistical analysis was used to determine the intra-individual biological variation of sweat chloride in each group of subjects.