Background and Objectives: The diaphragm, the lumbar multifidus muscles, and the thoracolumbar fascia (TLF) execute an important role in the stability of the lumbar spine and their morphology has been modified in subjects with non-specific low back pain (NS-LBP). While it is true that three structures correlate anatomically, the possible functional correlation between them has not been investigated previously in healthy subjects nor in subjects with NS-LBP. The aim of the present study was to examine this functional nexus by means of a comparison based on ultrasonographic parameters of the diaphragm, the lumbar multifidus muscles, and the TLF in subjects with and without NS-LBP. Materials and Methods: A sample of 54 (23 NS-LBP and 31 healthy) subjects were included in the study. The thickness and diaphragmatic excursion at tidal volume (TV) and force volume (FV), the lumbar multifidus muscles thickness at contraction and at rest, and the TLF thickness were evaluated using rehabilitative ultrasound imaging (RUSI) by B-mode and M-mode ultrasonography. The diaphragm thickening capacity was also calculated by thickening fraction (TF) at tidal volume and force volume. Results: There were no significant differences recorded between the activation of the diaphragm and the activation of the lumbar multifidus muscles and TLF for each variable, within both groups. However, there were significant differences recorded between both groups in diaphragm thickness and diaphragm thickening capacity at tidal volume and force volume. Conclusions: Diaphragmatic activation had no functional correlation with the activation of lumbar multifidus muscles and TLF for both groups. Nevertheless, subjects with NS-LBP showed a reduced diaphragm thickness and a lower diaphragm thickening capacity at tidal volume and force volume, compared to healthy subjects.