The passive elastic modulus of muscle fiber appears to be size-dependent. The objectives of this study were to determine whether this size effect was evident in the mechanical testing of muscle fiber bundles and to examine whether the muscle fiber bundle cross-section is circular. Muscle fibers and fiber bundles were extracted from lumbar spine multifidus and longissimus of three cohorts: group one (G1) and two (G2) included 13 (330 ± 14 g) and 6 (452 ± 28 g) rats, while Group 3 (G3) comprised 9 degenerative spine patients. A minimum of six muscle fibers and six muscle fiber bundles from each muscle underwent cumulative stretches, each of 10% strain followed by 4 minutes relaxation. For all specimens, top and side diameters were measured. Elastic modulus was calculated as tangent at 30% strain from the stress–strain curve. Linear correlations between the sample cross sectional area (CSA) and elastic moduli in each group were performed. The correlations showed that increasing specimen CSA resulted in lower elastic modulus for both rats and humans, muscle fibers and fiber bundles. The median ratio of major to minor axis exceeded 1.0 for all groups, ranging between 1.15–1.29 for fibers and 1.27–1.44 for bundles. The lower elastic moduli with increasing size can be explained by relatively less collagenous extracellular matrix in the large fiber bundles. Future studies of passive property measurement should aim for consistent bundle sizes and measuring diameters of two orthogonal axes of the muscle specimens.