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Background: It is unknown if serum concentrations of cobalamin, folate, canine pancreatic lipase immunoreactivity (cPLI), and canine trypsin-like immunoreactivity (cTLI) obtained postprandially are equivalent to measurements obtained after withholding food in dogs with suspected gastrointestinal disease., Hypothesis/objectives: Measurements of serum concentrations of cobalamin, folate, cPLI, and cTLI postprandially will be equivalent to measurements after 12 hours of withholding food in dogs with signs of chronic gastrointestinal disease. Changes observed will not alter clinical interpretation., Animals: 51 client-owned dogs with signs of gastrointestinal disease., Methods: Prospective single arm clinical trial. Serum concentrations of cobalamin, folate, cPLI and cTLI 2, 4, and 8 hours postprandially were compared by equivalence testing to values after withholding food for 12 hours (baseline)., Results: Mean serum cobalamin concentrations 2 hours (498.1 ± 213.1 ng/L; P = 0.024) and 4 hours (501.9 ± 207.4 ng/L; P = 0.008) postprandial were equivalent to baseline (517.3 ± 211.5 ng/L). Mean serum cTLI 2 hours (31.3 ± 14 μg/L; P < 0.001) and 4 hours (29.6 ± 13.1 μg/L; P = 0.027) postprandial were equivalent to baseline (31.1 ± 15 μg/L). Mean serum folate concentration 2 hours postprandial (15 ± 7.7 μg/L) was equivalent to baseline (13.7 ± 8.3 μg/L; P < 0.001). Equivalence could not be assessed for cPLI due to results below the lower limit of quantification. Feeding altered the clinical interpretation in 27% (cobalamin), 35% (folate), 20% (cTLI), and 12% (cPLI) of dogs., Conclusions and Clinical Importance: The clinical interpretation for a substantial number of samples changed after feeding, therefore withholding food before sample collection is prudent., (© 2024 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC on behalf of American College of Veterinary Internal Medicine.)