Abstract  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 (Δ17 ± 16 nmol (120 min) l⁻¹; P= 0.002) whereas there was no change in the SUR+EX group (Δ1 ± 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.