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Maternal diet may influence offspring's health, even within well-nourished populations. Amniotic fluid (AF) provides a rational compartment for studies on fetal metabolism. Evidence in animal models indicates that maternal diet affects AF metabolic profile; however, data from human studies are scarce. Therefore, we have explored whether AF content may be influenced by maternal diet, using a validated food-frequency questionnaire and implementing NMR-based metabolomics. Sixty-five AF specimens, from women undergoing second-trimester amniocentesis for prenatal diagnosis, were analysed. Complementary, maternal serum and urine samples were profiled. Hierarchical cluster analysis identified 2 dietary patterns, cluster 1 (C1, n = 33) and cluster 2 (C2, n = 32). C1 was characterized by significantly higher percentages of energy derived from refined cereals, yellow cheese, red meat, poultry, and "ready-to-eat" foods, while C2 by higher (P < 0.05) whole cereals, vegetables, fruits, legumes, and nuts. 1 H NMR spectra allowed the identification of metabolites associated with these dietary patterns; glucose, alanine, tyrosine, valine, citrate, cis-acotinate, and formate were the key discriminatory metabolites elevated in C1 AF specimens. This is the first evidence to suggest that the composition of AF is influenced by maternal habitual dietary patterns. Our results highlight the need to broaden the knowledge on the importance of maternal nutrition during pregnancy.