Lower back pain (LBP) has been the leading cause of disability since 1990. Objectives: The main objective of this observational case-control study was to evaluate, using ultrasound, whether there were differences in the onset and ratio of core muscle contraction between subjects with non-specific chronic lower back pain and healthy subjects. Methods: A total of 60 participants (52% women), split between those with non-specific chronic lower back pain (n = 26) and healthy (n = 34) subjects, were recruited. Initial muscle contraction of the lateral abdominal wall, pelvic floor, lumbar multifidus, and respiratory diaphragm was measured using ultrasound. The abdominal drawing-in maneuver, contralateral arm elevation, the Valsalva maneuver, and voluntary contraction of the pelvic floor in seated and standing positions were performed. The muscle thickness of the lateral abdominal wall and lumbar multifidus and excursion of the pelvic floor and diaphragm at rest and during testing were also analyzed. Results: No differences were found between the groups in the initial contraction. Statistically significant differences were found in the following variables: diaphragm excursion (p = 0.032, r = 0.277) and lumbar multifidus ratio (p = 0.010, r = 0.333) in the standing-abdominal retraction maneuver; pelvic floor excursion (p = 0.012, r = 0.325) in the standing-contralateral arm raise; and transverse abdominis ratio (p = 0.033, r = 0.275) in the sitting-contralateral arm raise. A statistically significant interaction between the groups and body mass index was observed in resting diaphragm excursion (p = 0.018, partial eta squared = 0.096) during sitting-voluntary pelvic floor contraction. Conclusions: It cannot be concluded that there is a specific pattern of core activation in any of the groups. However, statistically significant differences were found in the contraction indexes of the lumbopelvic musculature.