Introduction Little is known about the underlying biomechanical cause of low back pain (LBP). Recently, technological advances have made it possible to quantify biomechanical and neurophysiological measurements, potentially relevant factors in understanding LBP etiology. However, few studies have explored the relation between these factors. This study aims to quantify the correlation between biomechanical and neurophysiological outcomes in non-specific LBP and examine whether these correlations differ when considered regionally vs. segmentally. Methods This is a secondary cross-sectional analysis of 132 participants with persistent non-specific LBP. Biomechanical data included spinal stiffness (global stiffness) measured by a rolling indenter. Neurophysiological data included pain sensitivity (pressure pain threshold and heat pain threshold) measured by a pressure algometer and a thermode. Correlations were tested using Pearson’s product-moment correlation or Spearman’s rank correlation as appropriate. The association between these outcomes and the segmental level was tested using ANOVA with post-hoc Tukey corrected comparisons. Results A moderate positive correlation was found between spinal stiffness and pressure pain threshold, i.e., high degrees of stiffness were associated with high pressure pain thresholds. The correlation between spinal stiffness and heat pain threshold was poor and not statistically significant. Aside from a statistically significant minor association between the lower and the upper lumbar segments and stiffness, no other segmental relation was shown. Conclusions The moderate correlation between spinal stiffness and mechanical pain sensitivity was the opposite of expected, meaning higher degrees of stiffness was associated with higher pressure pain thresholds. No clinically relevant segmental association existed.