The mechanisms underlying pain relief following spinal manipulative therapy (SMT) are not understood fully although biomechanical and neurophysiological processes have been proposed. As such, we designed this randomized trial to elucidate the contributions of biomechanical and neurophysiological processes. A total of 132 participants with low back pain were randomly assigned to receive SMT at either the lumbar segment measured as the stiffest or the segment measured as having the lowest pain threshold. The primary outcome was patient reported low back pain intensity following treatment. Secondary outcomes were biomechanical stiffness and neurophysiological pressure pain threshold. All outcomes were measured at baseline, after the fourth and final session and at 2-weeks follow-up. Data were analyzed using linear mixed models, and demonstrated that the SMT application site did not influence patient reported low back pain intensity or stiffness. However, a large and significant difference in pressure pain threshold was observed between groups. This study provides support that SMT impacts neurophysiological parameters through a segment-dependent neurological reflex pathway, although this do not seem to be a proxy for improvement. This study was limited by the assumption that the applied treatment was sufficient to impact the primary outcome. Treatment of low back pain. Low back pain (LBP) is now the number one cause for years lived disability worldwide 1. In most cases, a specific pathoanatomical cause of LBP cannot be identified 2. Without a specific therapeutic target, a predictably large and diverse spectrum of interventions are available to clinicians that range from joint mobilization to spinal fusion surgery 3. Given these almost endless possibilities, clinical guidelines rate education and exercise as first line therapy for low back pain often in combination with manual therapy 3. Although, these guideline recommendations are generally clear and unambiguous, it is challenging for clinicians to implement them in practice (e.g. which exercises to recommend, how often, and which patients to offer manual therapy etc.). Spinal manipulative therapy. Spinal manipulative therapy (SMT) is a manual therapy recommended as a second line intervention for LBP in most clinical guidelines 4. However, like other conservative treatments, there is little evidence or consensus regarding the specifics of SMT application such as which patients are likely to respond, which type of SMT should be used, and which dose/frequency of SMT is optimal. While the specific SMT technique does not seem to be important 5-7 , there are at least two theoretical rationales for where to apply SMT: at the site of greatest biomechanical dysfunction or the site of greatest pain sensitivity. As the goal of SMT is to restore normal function to segments with biomechanical dysfunctions 8 , it may be surprising to some that the evidence for identifying such dysfunction is sparse. A narrative review reported that clinicians use a variety of different ways to ...