The purpose of this study was to investigate the differentiation in muscle tissue characteristics and recruitment between the deep and superficial multifidus muscle by magnetic resonance imaging. The multifidus is a very complex muscle in which a superficial and deep component can be differentiated from an anatomical, biomechanical, histological and neuromotorial point of view. To date, the histological evidence is limited to low back pain patients undergoing surgery and cadavers. The multifidus muscles of 15 healthy subjects were investigated with muscle functional MRI. Images were taken under three different conditions: (1) rest, (2) activity without pain and (3) activity after experimentally induced low back muscle pain. The T2 relaxation time in rest and the shift in T2 relaxation time after activity were compared for the deep and superficial samples of the multifidus. At rest, the T2 relaxation time of the deep portion was significantly higher compared to the superficial portion. Following exercise, there was no significant difference in shift in T2 relaxation time between the deep and superficial portions, and in the pain or in the non-pain condition. In conclusion, this study demonstrates a higher T2 relaxation time in the deep portion, which supports the current assumption that the deep multifidus has a higher percentage of slow twitch fibers compared to the superficial multifidus. No differential recruitment has been found following trunk extension with and without pain induction. For further research, it would be interesting to investigate a clinical LBP population, using this non-invasive muscle functional MRI approach.