Oral gavage is commonly used in pre-clinical drug evaluation, but is potentially aversive and may induce behavioral effects independent of compounds under investigation. This study examined the combined effects of repeated oral gavage and disease induction on anxiety-like behavior in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis. The C57BL/6J and NOD/ShiLtJ EAE variants were exposed to sham-EAE induction or untreated control conditions, and either daily oral gavage or home cage conditions. Anxiety-like behavior was subsequently assessed in the elevated plus maze. C57BL/6J mice exhibited increased anxiety-like behavior, relative to NOD/ShiLtJ mice, in response to repeated gavage, whereas sham-EAE induction and repeated gavage were associated with increased anxiety-like behavior in NOD/ShiLtJ mice. Thus, exposure to the induction procedure and repeated gavage differentially altered subsequent anxiety-like behavior in the two EAE variants. Future pre-clinical studies should rely on prior evaluation of parameters of the experimental design using sham-EAE mice. Additionally, less aversive administration routes should be utilized wherever possible to ensure that procedures do not distort effects of the therapeutic under investigation.