National Institute of Occupational Safety and Health, Japan Step Test (JST) is a valid approach for cardiorespiratory fitness assessment among subjects aged 30-60 years. It estimates maximal oxygen uptake (VO2max) based on the heart rate (HR) index, a combination of HRs during and after exercise. This study assessed the cross-validity of the VO2max estimation using the JST among adolescent university students. A total of 20 healthy male university students were included in this study. Measurements were taken over three days, with cardiopulmonary exercise testing, JST, and the Chester step test (CST) measured on each day. Pearson’s product-rate correlation coefficient or Spearman’s rank-sum correlation coefficient was used for examining the association between measured VO2max and estimated VO2max and between measured VO2max and variables in the JST equation. Bland-Altman analysis was also used to examine the presence and extent of systematic errors between measured and estimated VO2max. Estimated VO2max by the JST showed a stronger correlation with measured VO2max than estimated VO2max by the CST. Moreover, the HR index showed the strongest correlation with measured VO2max in variables of the JST equation. However, estimated VO2max by the JST showed fixed and proportional errors when compared to measured VO2max. It was suggested that the HR index, a combination of HRs during and after exercise, was useful for estimating VO2max among adolescent university students. Estimated VO2max using the JST showed a strong correlation with measured VO2max, however, some estimation accuracy problems such as systematic errors.