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Background/Objectives The paediatric reference range of fecal calprotectin (FC) has not been decisively established and previous studies show a wide within-age variability, suggesting that other factors like anthropometric data or type of feeding can influence FC. Our aims were to establish the normal levels of FC in healthy children grouped by age and analyze whether sex, gestational age, birth weight, type of delivery, type of feeding, or anthropometric data influence FC values. Methods This multicentre, cross-sectional, and observational study enrolled healthy donors under 18 years of age who attended their Primary Health Care Centre for their routine Healthy Child Program visits. The exclusion criteria were: (i) immunodeficiency, (ii) autoimmune or (iii) gastrointestinal disease; (iv) medication usage; (v) gastrointestinal symptoms; or (vi) positive finding in the microbiological study. Results We enrolled 395 subjects, mean age was 4.2 years (range 3 days to 16.9 years), and 204 were male. The median FC was 77.0 mcg/g (interquartile range 246). A negative correlation between age and FC was observed (Spearman's rho = −0.603, P<0.01), and none of the other factors analyzed were found to influence FC levels. Conclusions Normal FC values in healthy children (particularly in infants) are higher than those considered to be altered in adults and show a negative correlation with age. It is necessary to reconsider the upper limits of FC levels for paediatric patients according to age, with further studies required to determine other factors that influence FC during infancy. [ABSTRACT FROM AUTHOR]