The present investigation was undertaken to examine whether maximal oxygen uptake (VO2max) and anaerobic threshold (AT) measured during incremental treadmill exercise would be affected by the exercise protocol in trained and untrained individuals. Fifteen untrained men, 10 untrained women, and 12 trained individuals participated in this study. The Astrand, Bruce, and Costill/Fox protocols were selected for comparison. Each subject was tested using all three protocols and the three tests were conducted in a randomized counterbalanced order. During each test, oxygen uptake was measured every 30 s and the test was terminated according to the standard criteria. The VO2max was determined by averaging the two consecutive highest measurements, whereas AT was determined using ventilatory parameters following the V-slope technique. The Astrand, Bruce, and Costill/Fox protocols produced test durations of 9.8 (SEM 0.5), 12.4 (SEM 0.4), and 4.9 (SEM 0.3) min, respectively, in the untrained men, 9.0 (SEM 0.8), 11.0 (SEM 0.6), and 5.3 (SEM 0.6) min, respectively, in the untrained women, and 14.5 (SEM 0.5), 17.0 (SEM 0.5) and 10.4 (SEM 0.4) min, respectively, in the trained men. In the untrained men and women, no differences in VO2max were observed among the three different protocols, but AT was lower when using the Bruce compared to the Astrand protocol. In the trained men, VO2max and AT were lower when using the Bruce protocol than either the Astrand or Costill/Fox protocols. In conclusion, VO2max measured during treadmill exercise is not affected by the protocol of the test and using a running protocol of short duration (i.e. about 5 min) could be a time-efficient way of assessing VO2max in healthy untrained subjects. In trained subjects, however, a protocol consisting of running with small increments in gradient is effective in eliciting a higher VO2max. The lower AT associated with the Bruce protocol seen in both untrained and trained groups suggests this aerobic parameter is protocol dependent and this protocol dependency is not affected by training status.