Control of oxygen uptake (VO2) kinetics in skeletal muscle is mediated through interactions among ADP feedback, oxygen and substrate delivery, and mitochondrial enzyme activity. At the onset of contractions VO2 kinetics are well modelled by a mono-exponential function, consistent with a first-order control mechanism (1). Importantly, however, this model includes a time delay (δ) to account for the transit delay between the muscle capillary and the downstream site of the VO2 measurement (2). However, δVO2 may also include the influence of an allosteric control mechanism(s) in activating VO2 (2-4). In order to elucidate the nature of the VO2 time delay we aimed to determine δVO2 and the time delay in the change in deoxygenated hemoglobin and myoglobin (δ[ΔHHbMb]) during contractions in canine muscle with constant (pump) perfusion, before and after prior contractions to activate allosteric control processes. Nine dogs were anaesthetised with 30 mg.kg-1 pentobarbital (IV), and a deep surgical plane of anaesthesia was maintained with additional doses. Dogs were mechanically ventilated and the gastrocnemius and tendon were isolated and attached to a force transducer. A constant (high) blood flow was maintained by a pump during contractions before (S1) and after 3 min of priming contractions (PS1) separated by 2 min rest. Isometric tetanic contractions (50Hz; 200 ms duration) were elicited via supramaximal sciatic nerve stimulation at 0.33 Hz for 3 min. Muscle VO2 was determined contraction-by-contraction using an ultrasonic flowmeter and inline venous oximetry. Muscle [ΔHHbMb] was determined by near infrared spectroscopy. The kinetics of the fundamental VO2 and [ΔHHbMb] responses were modelled with a mono-exponential function. Blood flow was 1.04±0.20 L.kg-1.min-1 (mean±SD) across the rest-exercise transition in both conditions. δ[ΔHHbMb] was greater in S1 (3.5±1.4 s) than PS1 (1.8±1.8s; p=0.027). Similarly, δVO2 was greater in S1 (7.1±2.2 s) than PS1 (3.2±1.7s; p<0.001), which represented an apparent blood volume of 9.7±1.9 and 4.4±2.2 mL (p<0.001) in S1 and PS1, respectively. The VO2 amplitude and mean response time were 91.5±33.8 vs. 85.7±20.1 mL.min-1.kg-1 (p>0.05) and 18.4±4.8 vs. 21.0±4.2 s (p>0.05) in S1 and PS1 respectively. A first-order control mechanism suggests that, during constant blood flow, the time delay between the site of muscle gas exchange and the inline oximetry measurement should be constant. The reduction in δVO2 following prior contractions is not consistent with first-order control – rather allosteric features in the control of VO2 kinetics are implicated (4). That the δ[ΔHHbMb] was also reduced by prior contractions during constant perfusion supports this notion. In conclusion, a first-order control mechanism does not appear sufficient to explain the behaviour of the δVO2 at the onset of stimulated contractions in situ.