Freely moving pupillometry and awake vestibular recordings capture acute vestibular-autonomic signatures of motion sickness in mice.

Motion sickness (MS) in rodents is commonly inferred from behavioral and autonomic readouts, but objective measures capturing ocular and vestibular involvement remain limited. Here we implemented a multimodal framework combining freely moving pupillometry, awake vestibulo-ocular reflex (VOR), awake ocular vestibular evoked myogenic potentials (oVEMPs), and behavioral assays to characterize MS-like responses in mice. We further applied vestibular habituation training (VHT) to test whether these endpoints track adaptive recovery. Rotational stimulation induced a reproducible MS-like phenotype, including locomotor suppression, reduced feeding, and rapid hypothermia. Pupils dilated and ocular motility metrics decreased, yielding a reproducible ocular signature during vestibular conflict. Mydriasis coincided with robust c-Fos induction in the locus coeruleus (LC). Horizontal VOR gain decreased at low-mid stimulus frequencies, and oVEMPs showed reduced amplitude with prolonged latency, indicating dissociable vestibular readouts following rotation. After 14 days of VHT, behavioral, ocular, and vestibular measures largely returned toward control levels, consistent with adaptive recalibration. Together, this framework provides physiologically grounded endpoints beyond classical behavioral assays and may facilitate more precise characterization of vestibular conflict and its adaptive resolution in preclinical studies.