Background: The purpose of this study was to determine if mixing of fluoroquinolones with a common enteral feeding formulation, Ensure (Ross Products Division, Abbott Laboratories, Columbus, OH), would alter the measured in vitro quinolone concentrations over a 24-hour period. Methods: Tablets of ciprofloxacin (500 mg), levofloxacin (500 mg), and ofloxacin (300 mg) were crushed and mixed with 240 mL of Ensure, water and calcium chloride (500 mg/L), water and magnesium chloride (200 mg/L), water and calcium chloride and magnesium chloride, and water alone. Fluoroquinolone concentrations of the mixtures were measured, via high-performance liquid chromatography, at baseline and serially over 24 hours. Experiments were performed in duplicate, at three temperatures (5°C, 25°C, and 37°C). Results: Average decreases of 82.5% ± 1.5% for ciprofloxacin, 61.3% ± 5.2% for levofloxacin, and 45 7% ± 10.1% for ofloxacin (mean ± 95% CI) were observed in vitro for Ensure over the two experimental sets at baseline. Serial analysis revealed no further significant change in any of the quinolone concentrations over the remaining 24-hour period. No significant decrease was noted with the quinolones when mixed in water and calcium, water and magnesium, water and calcium and magnesium, or water alone. This phenomenon appears to be unaffected by time and temperature. Conclusions: These data suggest there is an immediate and significant loss of fluoroquinolone when mixed with Ensure. An explanation for the loss of fluoroquinolone remains unclear.