Although several mechanical devices have been developed to objectively assess posteroanterior (PA) stiffness of the lumbar spine, no standardized testing protocol has been adopted. Two factors that may vary across protocols, and that effect measured stiffness and the comfort of the test subject, are the size of the indenter head used to apply the PA pressure, and indenting frequency. Three variables; PA stiffness, defined as the slope of the stiffness curve (K), the displacement of the indenter at 30N (D30), and rating of perceived comfort, were measured in 36 subjects asymptomatic for low back pain. For each subject nine tests were conducted, using three different indenter head sizes (300 mm2, 720 mm2 and 1564 mm2) at each of three different testing frequencies (0.25 Hz, 0.5 Hz and 2 Hz). Machine testing with a large indenter head produced a lower K value, an increased D30 value and higher perceived comfort, while a fast testing frequency produced a higher K value and a lower D30 value. An indenter size by frequency interaction showed small indenter heads to be least comfortable at slow speed. The differences found suggest that the indenter head size and the testing frequency should be standardized during mechanical spinal stiffness testing.