Background <p>The effect of the simultaneous administration of local anesthetics, whether synergistic, additive, or antagonistic, has not yet been established. This study investigated the interaction between concurrently administered short- and long-acting local anesthetics.</p> Methods <p>A total of 160 male Sprague–Dawley rats were divided into 16 groups (<i>n</i> = 10 each): 4 local anesthetic alone groups (lidocaine, mepivacaine, ropivacaine, and levobupivacaine) and 12 simultaneous administration groups that received adjusted concentrations of short- and long-acting local anesthetics. Local anesthetics were administered at a constant rate until seizure onset, which was determined by electroencephalographic monitoring. The cumulative dose required to induce seizures was calculated in each group. A linear regression analysis was performed to assess the relationship between the doses of the combined agents, with additivity inferred from an adjusted R² of &gt; 0.7. In addition, pharmacodynamic interactions were evaluated using an interaction index based on the Loewe additivity model.</p> Results <p>For all combinations of local anesthetics, R² was &gt; 0.7. A linear regression analysis indicated an additive effect for each combination. Furthermore, an analysis of the interaction index indicated a tendency toward additivity.</p> Conclusions <p>This study revealed no clear evidence of antagonism or synergism for the simultaneous administration of short-acting (lidocaine or mepivacaine) and long-acting anesthetics (ropivacaine or levobupivacaine). An additive effect was suggested in terms of the seizure threshold when short- and long-acting local anesthetics were administered simultaneously. This finding may therefore support the safe and effective combined use of local anesthetics in clinical regional anesthesia.</p>