Introduction In this prospective randomized trial, we examined the effect of three popular exercise training modalities on maximal oxygen uptake (VO2max) in overweight and obese individuals. In addition, we examined possible concomitant adaptations in endurance exercise performance (time to exhaustion (TTE)), citrate synthase (CS) activity, venous and arterial function, blood volume, and calculated stroke volume (SV). Methods Thirty subjects were recruited (age, 41 +/- 9 yr; weight, 91 +/- 14 kg; height, 173 +/- 8 cm; body mass index, 30 +/- 4 kg center dot m(-2)) and randomized to either 6 wk of 4 x 4-min high-intensity interval training (4HIIT) at 85%-95% of HRmax, 10 x 1-min HIIT (1HIIT) at VO2max load, or 45-min moderate-intensity continuous training (MICT) at 70% of HRmax. VO2max, TTE, CS activity, venous and arterial function, as well as blood volume were measured before and after the training period. O-2 pulse was calculated and used to estimate SV. Analysis was conducted per protocol. Results Only 4HIIT increased VO2max (P < 0.01) and significantly more compared with 1HIIT (P = 0.04) and MICT (P = 0.03) (4HIIT, 10%; 1HIIT, 3.3%; and MICT, 3.1%). All groups increased TTE (4HIIT, 198%; 1HIIT, 116%; MICT, 52%), with a higher increase after 4HIIT compared with that after MICT (P = 0.02). Calculated SV increased only after 4HIIT (14.4%). Plasma volume and hemoglobin mass increased after 1HIIT only (5.6% and 6.5%); however, no group differences were found. All groups increased CS activity (4HIIT, 35%; 1HIIT, 35%; MICT, 56%), with no group differences. Arterial inflow (15.7%) and venous outflow (22.7%) decreased after MICT, but there were no group differences. Conclusions 4HIIT was superior to 1HIIT and MICT in improving VO2max likely because of an increased SV.