The primary objective of this study was to investigate the effects of step height on the Physical Efficiency Index (PEI) of a three-minute step test. The subjects were divided into three groups according to their heights (159-168 cm, 169-178 cm, and 179-188 cm), with 10 subjects in each group. All the subjects performed the three-minute step test with two different step heights (a 35 cm step height and a step height adjusted to the height of the tibial tuberosity). The i-FITT-HRM2 instrument developed by Microtime Computer Inc. was used to monitor the subject’s heart rate variation. Data were analyzed using Welch/ Brown Forsythe test and paired sample t-tests to investigate the effects caused by the difference in step height. The results were followed: (1) There was no significant difference in the PEI in the three-minute step test performed at the 35 cm step height (p＞.01). (2) There was also no significant difference in the PEI in the three-minute step test performed at the adjusted step height (p＞.01). (3) Subjects in all three groups exhibited lower PEIs when undergoing the three-minute step test performed at the adjusted step height compared to that at the 35 cm step height, where the 169-178 cm group and the 179-188 cm group exhibited significant differences in this respect (p ＜.01). The conclusion of this study were that the group of subjects with body heights between 159 cm and 168 cm demonstrated no significant difference after undergoing the three-minute step test at a step height of 35 cm and a step height adjusted to the height of the tibial tuberosity. Hence, it is believed that the test performed at a 35 cm step height is suitable for subjects with body heights between 159 cm and 168 cm. The other two groups exhibited significant differences between the three-minute step tests performed at the 35 cm step height and the adjusted step height, i.e., a decline in PEI after step height adjustment. Therefore, the appropriate step height for these two groups of subjects requires further investigation.