BackgroundIt 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/ObjectivesMeasurements 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.Animals51 client‐owned dogs with signs of gastrointestinal disease.MethodsProspective 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).ResultsMean 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 ImportanceThe clinical interpretation for a substantial number of samples changed after feeding, therefore withholding food before sample collection is prudent.