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BACKGROUND: Ground-based studies have reported shifts of the vestibulo-ocular reflex (VOR) slow phase velocity (SPV) axis toward the resultant gravito-inertial force vector. The VOR was examined during eccentric roll rotation before, during and after an 8-day orbital mission. On orbit this vector is aligned with the head z-axis. Our hypothesis was that eccentric roll rotation on orbit would generate horizontal eye movements. METHODS: Two subjects were rotated in a semi-supine position with the head nasal-occipital axis parallel to the axis of rotation and 0.5 m off-center. The chair accelerated at 120 deg/s2 to 120 deg/s, rotated at constant velocity for one minute, and then decelerated to a stop in similar fashion. RESULTS: On Earth, the stimulation primarily generated torsional VOR. During spaceflight, in one subject torsional VOR became horizontal VOR, and then decayed very slowly. In the other subject, torsional VOR was reduced on orbit relative to pre- and post-flight, but the SPV axis did not rotate. CONCLUSION: We attribute the shift from torsional to horizontal VOR on orbit to a spatial orientation of velocity storage toward alignment with the gravito-inertial force vector, and the inter-individual difference to cognitive factors related to the subjective straight-ahead. [ABSTRACT FROM AUTHOR]