The objectives of the study were to investigate changes in pain perception and neural activity during tonic pain due to altered sensory input from the spine following chiropractic spinal adjustments. Fifteen participants with subclinical pain (recurrent spinal dysfunction such as mild pain, ache or stiffness but with no pain on the day of the experiment) participated in this randomized cross-over study involving a chiropractic spinal adjustment and a sham session, separated by 4.0 ± 4.2 days. Before and after each intervention, 61-channel electroencephalography (EEG) was recorded at rest and during 80 seconds of tonic pain evoked by the cold-pressor test (left hand immersed in 2 °C water). Participants rated the pain and unpleasantness to the cold-pressor test on two separate numerical rating scales. To study brain sources, sLORETA was performed on four EEG frequency bands: delta (1-4 Hz), theta (4-8 Hz), alpha (8-12 Hz) and beta (12-32 Hz). The pain scores decreased by 9% after the sham intervention (p < 0.05), whereas the unpleasantness scores decreased by 7% after both interventions (p < 0.05). sLORETA showed decreased brain activity following tonic pain in all frequency bands after the sham intervention, whereas no change in activity was seen after the chiropractic spinal adjustment session. This study showed habituation to pain following the sham intervention, with no habituation occurring following the chiropractic intervention. This suggests that the chiropractic spinal adjustments may alter central processing of pain and unpleasantness.