Background: The aim of the current study was to examine different features of the rectus abdominis muscle (RA) in patients with and without a midline incisional hernia to characterize the effects of a hernia on abdominal wall skeletal muscle. Material and methods: RA tissue from patients undergoing surgical repair of a large midline incisional hernia (n = 18) was compared with that from an intact abdominal wall in patients undergoing colorectal resection for benign or low-grade malignant disease (n = 18). In addition, needle biopsies were obtained from the vastus lateralis muscle (VL) of all subjects. Outcome measures were muscle fiber type and size, preoperative truncal flexion strength and leg extension power measured in strength-measure equipment, and RA cross-sectional area measured by computed tomography. Results: In both the RA and VL, the fiber cross-sectional area was greater in the patients with a hernia. The RA cross-sectional area correlated significantly with the truncal flexion strength (r = 0.44, P = 0.015). Patients in the hernia group had a significantly reduced ratio between truncal flexion strength and RA cross-sectional area compared with the control group (41.3 ± 11.5 N/cm² versus 51.2 ± 16.3 N/cm², P = 0.034). Conclusions: Anatomical displacement of the RA and lack of medial insertion in the linea alba rather than dysfunction secondary to alteration of muscle fiber structure may contribute to impairment of abdominal wall function in patients with midline incisional hernias. The study was registered at http://www.clinicaltrials.gov/(NCT02011048).