OBJECTIVE To evaluate the effects of withholding food on the results for measurements of serum concentrations of cobalamin, folate, canine pancreatic lipase immunoreactivity (cPLI), and canine trypsin-like immunoreactivity (cTLI) in healthy dogs. ANIMALS 11 healthy employee- or student-owned dogs. PROCEDURES Food was withheld from the dogs for 12 hours, baseline blood samples were collected, then dogs were fed. Postprandial blood samples collected 1, 2, 4, and 8 hours later were assessed. A mixed-effects ANOVA model with fasting duration (time) as a fixed factor and dog as a random effect was fit for each analyte variable. Additionally, a mixed-effects ANOVA model controlling for the variable of time was fit to assess whether lipemia affected serum concentrations of the analytes. RESULTS The median serum cobalamin concentration was lower at 4 hours (428 ng/L) and 8 hours (429 ng/L) postprandially, compared with baseline (479 ng/L), but this difference was not clinically meaningful. Although there were no substantial differences in serum concentrations of folate, cPLI, or cTLI, postprandial changes in serum concentrations of cTLI or folate could potentially affect diagnoses in some dogs. CONCLUSIONS AND CLINICAL RELEVANCE Although results indicated that feedings rarely resulted in clinically important differences in the median serum concentrations of cobalamin, folate, cPLI, or cTLI in healthy dogs, given the further processing required for lipemic samples, withholding food for at least 8 hours is an appropriate recommendation when measuring these analytes. Similar research is needed in dogs with gastrointestinal disease to determine whether the withholding of food is necessary when measuring these analytes in affected dogs.