OBJECTIVE: The objective was to study the in vivo effects of anesthetic agents on peripheral nerve excitability. METHODS: Normal male mice were anesthetized by either isoflurane inhalation or a combination of medetomidine, midazolam, and butorphanol intraperitoneal injection ("triple agents"). Immediately after induction, the tail sensory nerve action potential was recorded and its excitability was monitored. RESULTS: Under both anesthetic protocols, there was an interval excitability change by long hyperpolarizing currents. There was greater threshold reduction approximately 30min post induction, in comparison to immediately post induction. Other excitability parameters were stable over time. Modeling suggested interval suppression of internodal H conductance or leak current. CONCLUSIONS: Anesthetic agents affected responses to long hyperpolarizing currents. SIGNIFICANCE: Axonal excitability during intraoperative monitoring may be affected by anesthetic agents. Interpretation of interval excitability changes under anesthesia requires caution, especially with long hyperpolarizing currents.