Key points Mitochondrial respiratory sensitivity to ADP is thought to influence muscle fitness and is partly regulated by cytosolic–mitochondrial diffusion of ADP or phosphate shuttling via creatine/phosphocreatine (Cr/PCr) through mitochondrial creatine kinase (mtCK). Previous measurements of respiration in vitro with Cr (saturate mtCK) or without (ADP/ATP diffusion) show mixed responses of ADP sensitivity following acute exercise vs. less sensitivity after chronic exercise. In human muscle, modelling in vivo ‘exercising’ [Cr:PCr] during in vitro assessments revealed novel responses to exercise that differ from detections with or without Cr (±Cr). Acute exercise increased ADP sensitivity when measured without Cr but had no effect ±Cr or with +Cr:PCr, whereas chronic exercise increased sensitivity ±Cr but lowered sensitivity with +Cr:PCr despite increased markers of mitochondrial oxidative capacity. Controlling in vivo conditions during in vitro respiratory assessments reveals responses to exercise that differ from typical ±Cr comparisons and challenges our understanding of how exercise improves metabolic control in human muscle. Abstract Mitochondrial respiratory control by ADP (Kmapp) is viewed as a critical regulator of muscle energy homeostasis. However, acute exercise increases, decreases or has no effect on Kmapp in human muscle, whereas chronic exercise surprisingly decreases sensitivity despite greater mitochondrial content. We hypothesized that modelling in vivo mitochondrial creatine kinase (mtCK)‐dependent phosphate‐shuttling conditions in vitro would reveal increased sensitivity (lower Kmapp) after acute and chronic exercise. The Kmapp was determined in vitro with 20 mm Cr (+Cr), 0 mm Cr (−Cr) or ‘in vivo exercising’ 20 mm Cr/2.4 mm PCr (Cr:PCr) on vastus lateralis biopsies sampled from 11 men before, immediately after and 3 h after exercise on the first, fifth and ninth sessions over 3 weeks. Dynamic responses to acute exercise occurred throughout training, whereby the first session did not change Kmapp with in vivo Cr:PCr despite increases in −Cr. The fifth session decreased sensitivity with Cr:PCr or +Cr despite no change in −Cr. Chronic exercise increased sensitivity ±Cr in association with increased electron transport chain content (+33–62% complexes I–V), supporting classic proposals that link increased sensitivity to oxidative capacity. However, in vivo Cr:PCr reveals a perplexing decreased sensitivity, contrasting the increases seen ±Cr. Functional responses occurred without changes in fibre type or proteins regulating mitochondrial–cytosolic energy exchange (mtCK, VDAC and ANT). Despite the dynamic responses seen with ±Cr, modelling in vivo phosphate‐shuttling conditions in vitro reveals that ADP sensitivity is unchanged after high‐intensity exercise and is decreased after training. These findings challenge our understanding of how exercise regulates skeletal muscle energy homeostasis.