Key points• Physical exercise significantly improves health but to what extent these benefits depend on altered energy balance remains unclear.• In a human experimental model, we investigated whether daily exercise could counteract the effects of short-term overfeeding and under-activity independent of its impact on energy imbalance in healthy young men.• Short-term positive energy balance from overfeeding and under-activity resulted in impaired metabolic outcomes and alterations in the expression of several key genes within adipose tissue involved in nutritional balance, metabolism and insulin action.• These changes were mostly prevented by the addition of a daily vigorous-intensity exercise bout even in the face of a standardised energy surplus.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 ( 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. Abbreviations AKT1, RAC-α serine/threonine-protein kinase; AKT2, RAC-β serine/threonine-protein kinase; ALT, alanine transaminase; AMPK, 5 AMP-activated protein kinase; CI, confidence interval; CRP, C-reactive protein; Ct, threshold cycle; DEXA, dual-energy X-ray absorptiometry; DIT, diet-induced thermogenesis; DNL, de novo lipogenesis; EPOC, excess post-exercise oxygen consumption; FAS, fatty acid synthase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GLUT4, glucose transporter type 4; HDL, high-density lipoprotein; HOMA-IR, homeostasis model assessment for insulin resistance; HOMA-β, homeostasis model assessment for...