The identification of relevant and valid biomarkers to distinguish patients with non-specific chronic low back pain (NSCLBP) from an asymptomatic population in terms of musculoskeletal factors could contribute to patient follow-up and to evaluate therapeutic strategies. Several parameters related to movement impairments have been proposed in the literature in that respect. However, most of them were assessed in only one study, and only 8% were evaluated in terms of reliability, validity and interpretability. The aim of this study was to consolidate the current knowledge about movement biomarkers to discriminate NSCLBP patients from an asymptomatic population. For that, an experimental protocol was established to assess the reliability, validity and interpretability of a set of 72 movement biomarkers on 30 asymptomatic participants and 30 NSCLBP patients. Correlations between the biomarkers and common patient reported outcome measures were also analysed. Four biomarkers reached at least a good level in reliability (ICC ≥ 0.75) and validity (significant difference between asymptomatic participants and NSCLBP patients, p ≤ 0.01) domains and could thus be possibly considered as valuable biomarkers: maximal lumbar sagittal angle, lumbar sagittal angle range of motion, mean lumbar sagittal angular velocity, and maximal upper lumbar sagittal angle during trunk sagittal bending. These four biomarkers demonstrated typically larger values in asymptomatic participants than in NSCLBP patients. They are in general weakly correlated with patient reported outcome measures, arguing for a potential interest in including related musculoskeletal factors in the establishment of a valuable diagnosis and in guiding treatment response.