Objective: To analyze the influence of dilution, time, and temperature after preparation on the osmolality of infant formulas given to newborns (NBs).Methods: Experimental and descriptive study with samples of different neonatal formulas to verify the osmolality of the milk according to dilution, time, and temperature after preparation. We analyzed seven neonatal formulas in the following times after preparation: immediately (up to 5 minutes); 20 and 40 minutes; every hour up to 8 hours; and 12 and 24 hours. The samples were evaluated at room temperature and after refrigeration. Osmolality curves were designed with the mean of triplicate samples of each milk sample. The digital Osmometer A+, model 3320, from Advanced Instruments measured the osmolality.Results: The time and temperature at which the milk was subjected after preparation did not cause the osmolality to exceed its safety cut-off point at a 1:30 dilution in any of the types of milk analyzed. At a 1:25 dilution, the formula with prebiotics in its composition went over the limit after 4 hours.Conclusions: The milk tested did not exceed the cut-off point of 450 mOsm/kg (approximately 400 mOsm/L), indicated as safe by the American Academy of Pediatrics (AAP) at a dilution recommended by manufacturers. It is important to know the factors that may or may not contribute to the rise of osmolality, in order to establish safe and quality practices for NBs, following protocols based on scientific evidence.