Rollover crashes are dynamic and complex events in which head impacts with the roof can cause catastrophic neck injuries. Ex vivo and computational models are valuable in understanding, and ultimately preventing, these injuries. Although neck posture and muscle activity influence the resulting injury, there is currently no in vivo data describing these parameters immediately prior to a head-first impact. The specific objectives of this study were to determine the in vivo neck vertebral alignment and muscle activation levels when upside down, a condition that occurs during a rollover. Eleven human subjects (6F, 5M) were tested while seated upright and inverted in a custom-built apparatus. Vertebral alignment was measured using fluoroscopy and muscle activity was recorded using surface and indwelling electrodes in 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 lordotic, C1 was aligned posterior to C7, the Frankfort plane was extended, and the activity of six muscles increased compared to upright and relaxed. When inverted subjects were asked to look forward to eliminate head extension, flexor muscle activity increased, C7 was more flexed, and C1 was aligned anterior to C7 versus upright and relaxed. Combined with the large inter-subject variability observed, these findings indicate that cadaveric or computational models designed to study injuries and prevention devices while inverted need to consider a variety of postures and muscle conditions to be relevant to the in vivo situation.