Purpose: Lumbar paraspinal muscle morphology has recently been evaluated in several studies with conflicting results. Several studies have performed single-slice evaluations of paraspinal muscle morphology, whereas other studies have done a multi-level assessment; this methodological difference might explain the observed different results. Our study evaluated if a single-slice axial measurement is representative of the entire lumbar musculature. Methods: We included 80 adult patients who were consecutively evaluated with magnetic resonance imaging (MRI) for spinal symptoms. Using T2-weighted axial images, we measured the fat signal fractions (FSF) and cross-sectional area (CSA) of the erector spinae and multifidus at the five levels of the lumbar spine (from L1-L2 to L5-S1). We used the ANOVA test for repeated measurements (with Bonferroni correction) to compare the FSF and CSA among the levels. Results: Erector spinae showed an increasing FSF from L1-L2 to L5-S1; all erector spinae FSF comparisons among the different levels were significantly different. Multifidus FSF also increased caudally below L2-L3, although significant differences were observed only with two or more levels of distance. The CSA of the erector spinae showed a caudal decrease (L4-L5 and L5-S1 being significantly smaller than all the levels above). The CSA of the multifidus showed that all levels exhibited a significantly different area compared to their adjacent level (except L5-S1 compared to L4-L5). Conclusions: No single-level FSF or CSA is representative of the whole lumbar spine. A standardized multi-level evaluation of the paraspinal musculature should be used in future research.