Serum cobalamin, folate, canine pancreatic lipase immunoreactivity (cPLI), and canine trypsin-like immunoreactivity (cTLI) are markers for canine small intestinal and exocrine pancreatic disease. Patients are often fasted prior to sampling to avoid theoretical postprandial effects. Guidance on how long food should be withheld is inconsistent, varying from 6 to 12 hours, and recommendations for withholding food are not evidence-based. This study compared serum levels of cobalamin, folate, cPLI, and cTLI in healthy dogs (n = 11) after 12 hours of being fasted, then 1, 2, 4, and 8 hours after being fed. Median serum cobalamin levels were mildly decreased 4 and 8 hours postprandially. Although these results were statistically significant, clinical diagnosis would not be altered. Differences in cPLI, cTLI, and folate were not significant. Postprandial lipemia did not affect serum concentrations of these markers when samples were adequately centrifuged, but withholding food for ≥8 hours was deemed useful for avoiding the centrifugation process required when samples were lipemic.