Objective-To determine whether feeding causes a change in feline trypsin-like immunoreactivity (fTLI) in serum from healthy cats. Animals-6 healthy domestic shorthair cats, Procedures-For the first 12 days of the study, 3 cats were fed a high-protein, high-fat (diet 1), and the other 3 were fed a maintenance (diet 2). On day 12, diets were switched, and cats were fed the other diet for the remaining 12 days of the study. On days 11 and 23, food was withheld for 24 hours, and baseline serum mi was measured. Cats were offered food equivalent to half their daily caloric maintenance requirements, and serum fTLI was measured 1, 2, 4, 6, 12, and 24 hours later. Uneaten food was removed after 1 hour. Results-Overall mean +/- SD serum fTLI was 22.7 +/- 5.8 mu g/L when cats were fed diet 1 and 21.1 +/- 5.0 mu g/L when cats were fed diet 2, There was no significant difference in serum fTLI over time or between diets. However, there was a statistically significant, but clinically unimportant (mean increase, 1.7 mu g/L), increase in serum fTLI, compared with baseline values, 1 hour after cats were fed diet 2 but not when cats were fed diet 1, Conclusions and Clinical Relevance-A maintenance diet may cause a clinically unimportant increase in serum fTLI 1 hour after feeding in healthy cats. Results suggest that for healthy cats, it is not necessary to withhold food before collecting samples for determination of ftLI in serum. Whether feeding changes fTLI in serum from cats with disorders of the exocrine portion of the pancreas remains to be determined.