Rollover accidents are dynamic and complex events in which head contacts with the roof can cause catastrophic injuries to the neck. To both study and ultimately prevent these injuries, the in vivo neck vertebral alignment and neck muscle activation levels immediately prior to a headfirst impact must be known. To our knowledge there is almost no data of this type available. Therefore, the aim of this study was to explore how occupants react to a headfirst impact scenario, such as a rollover, by altering their neck muscle activation and their neck alignment. The first step to advancing our understanding was to quantify which neck muscles are active and what the spinal posture is in a quasi-static upside-down posture. Six human subjects were tested while seated upright and inverted in a custom built roll apparatus. Vertebral alignment was measured using fluoroscopy and neck muscle activity was recorded using surface and fine- wire electrodes for eight superficial and deep neck muscles. In vivo vertebral alignment and muscle activation levels differed between the upright and inverted conditions. When inverted and relaxed, the neck was more curved and muscle activation was higher than when upright and relaxed. The results of this study provide an in vivo data set of vertebral and muscular response to an inverted configuration which can be used to improve and validate cadaveric and computational models and advance injury prevention strategies.