Physical activity can affect many aspects of metabolism but it is unclear to what extent this relies on manipulation of energy balance. Twenty-six active men age 25 ± 7 years (mean ± SD) were randomly assigned either to consume 50% more energy than normal by over-consuming their habitual diet for 7 days whilst simultaneously restricting their physical activity below 4000 steps day(-1) to induce an energy surplus (SUR group; n = 14) or to the same regimen but with 45 min of daily treadmill running at 70% of maximum oxygen uptake (SUR+EX group; n = 12). Critically, the SUR+EX group received additional dietary energy intake to account for the energy expended by exercise, thus maintaining a matched energy surplus. At baseline and follow-up, fasted blood samples and abdominal subcutaneous adipose tissue biopsies were obtained and oral glucose tolerance tests conducted. Insulinaemic responses to a standard glucose load increased 2-fold from baseline to follow-up in the SUR group (17 ± 16 nmol (120 min) l(-1); P = 0.002) whereas there was no change in the SUR+EX group (1 ± 6 nmol (120 min) l(-1)). Seven of 17 genes within adipose tissue were differentially expressed in the SUR group; expression of SREBP-1c, FAS and GLUT4 was significantly up-regulated and expression of PDK4, IRS2, HSL and visfatin was significantly down-regulated (P ≤ 0.05). The pAMPK/AMPK protein ratio in adipose tissue was significantly down-regulated in the SUR group (P = 0.005). Vigorous-intensity exercise counteracted most of the effects of short-term overfeeding and under-activity at the whole-body level and in adipose tissue, even in the face of a standardised energy surplus.