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Intraoperative auditory brainstem response (ioABR) testing following tympanostomy tube (TT) placement may be biased due to temporary threshold shifts (TTS). The purpose of the study was to assess the evidence for TTS in children who have undergone ioABR using prolonged latencies of wave I (males > 1.95 ms, females > 1.88 ms) as a marker of a persisting air–bone gap. Eighty-three children underwent ioABR following surgical procedures at University Hospital Bonn, Germany. The primary outcome measure was the latency of wave I at 80-dB SPL. The total sample consisted of 66 males (79.5%) and 17 females (20.5%) with a mean (SD) age of 46.4 (26.6) months. Of 163 operated ears (83 children), 72 (44.2%) had no middle ear fluid, 19 (11.6%) serous fluid, and 72 (44.2%) mucoid fluid. The risk of having a prolonged latency of wave I at 80-dB SPL was OR 4.61 (95% CI 2.01–10.59; p < 0.001) in those with mucoid fluid as compared to those without mucoid fluid. Intraoperative ABR results should account for sex differences and be interpreted with caution and be verified. Ultimately, parents should be engaged in a preoperative discussion to decide if an ioABR should be postponed if mucoid fluid was found.