Key points A slow adjustment of skeletal muscle oxygen uptake (V̇O2) to produce energy during exercise predisposes to early fatigue. In human studies, V̇O2 kinetics are slow when exercise is initiated from an elevated baseline; this is proposed to reflect slow blood flow regulation and/or recruitment of muscle fibres containing few mitochondria. To investigate this, we measured V̇O2 kinetics in canine muscle, with experimental control over muscle activation and blood flow. We found that V̇O2 kinetics remained slow when contractions were initiated from an elevated baseline despite experimentally increased blood flow and uniform fibre activation. These data challenge our current understanding of the control of muscle V̇O2 and demand consideration of new alternative mediators for V̇O2 control. Abstract Oxygen uptake kinetics (τV̇O2) are slowed when exercise is initiated from a raised metabolic rate. Whether this reflects the recruitment of muscle fibres differing in oxidative capacity, or slowed blood flow (Q̇) kinetics is unclear. This study determined τV̇O2 in canine muscle in situ, with experimental control over muscle activation and Q̇ during contractions initiated from rest and a raised metabolic rate. The gastrocnemius complex of nine anaesthetised, ventilated dogs was isolated and attached to a force transducer. Isometric tetanic contractions (50 Hz; 200 ms duration) via supramaximal sciatic nerve stimulation were used to manipulate metabolic rate: 3 min stimulation at 0.33 Hz (S1), followed by 3 min at 0.67 Hz (S2). Circulation was initially intact (SPON), and subsequently isolated for pump‐perfusion (PUMP) above the greatest value in SPON. Muscle V̇O2 was determined contraction‐by‐contraction using an ultrasonic flowmeter and venous oximeter, and normalised to tension‐time integral (TTI). τV̇O2/TTI and τQ̇ were less in S1SPON (mean ± s.d.: 13 ± 3 s and 12 ± 4 s, respectively) than in S2SPON (29 ± 19 s and 31 ± 13 s, respectively; P < 0.05). τV̇O2/TTI was unchanged by pump‐perfusion (S1PUMP, 12 ± 4 s; S2PUMP, 24 ± 6 s; P < 0.001) despite increased O2 delivery; at S2 onset, venous O2 saturation was 21 ± 4% and 65 ± 5% in SPON and PUMP, respectively. V̇O2 kinetics remained slowed when contractions were initiated from a raised metabolic rate despite uniform muscle stimulation and increased O2 delivery. The intracellular mechanism may relate to a falling energy state, approaching saturating ADP concentration, and/or slowed mitochondrial activation; but further study is required. These data add to the evidence that muscle V̇O2 control is more complex than previously suggested.