The purpose of this research was to develop the 3 min incremental step-in-place (3MISP) test for predicting maximal oxygen uptake (V•O2max). A total of 205 adults (20–64 years) completed the 3MISP and V•O2max tests. Using age, gender, body composition (BC) including percent body fat (PBF) or body mass index (BMI), and with or without heart rate (HR) at the beginning of exercise (HR0) or difference between HR at the third minute during the exercise and the first minute post exercise (ΔHR3 − HR4) in the 3MISP test, six V•O2max prediction models were derived from multiple linear regression. Age (r = −0.239), gender (r = 0.430), BMI (r = −0.191), PBF (r = −0.706), HR0 (r = −0.516), and ΔHR3 − HR4 (r = 0.563) were significantly correlated to V•O2max. Among the six V•O2max prediction models, the PBF model∆HR3 − HR4 has the highest accuracy. The simplest models with age, gender, and PBF/BMI explained 54.5% of the V•O2max in the PBF modelBC and 39.8% of that in the BMI modelBC. The addition of HR0 and ∆HR3 − HR4 increases the variance of V•O2max explained by the PBF and BMI models∆HR3 − HR4 by 17.98% and 45.23%, respectively, while standard errors of estimate decrease by 10.73% and 15.61%. These data demonstrate that the models established using 3MISP-HR data can enhance the accuracy of V•O2max prediction.