The force produced by the calf plantar flexors was measured according to Fisher & White (2003). Subjects were seated in a dynamometer with the lumbar spine against the back of the bench, the thigh of the preferred leg horizontal and the ankle at 85 deg (1.46 rad). A curved metal plate was clamped proximal to the knee joint, preventing heel lift during contraction. The upward force generated by the triceps surae was amplified and transmitted to an analog-to-digital converter (Cambridge Electronic Design 1401plus, CED, Cambridge, UK). The force produced was displayed on a chart recorder and recorded on Spike 2 computer software (Cambridge Electronic Design) at a frequency of 250 Hz. Heart rate (HR) was recorded using a three-lead ECG (Cardiorater CR7, Cardiac Records Ltd, London, UK), whilst blood pressure (BP) was monitored from the middle finger of the right hand using a Finapres system (Ohmeda 2300, Louisville, CO, USA). Whole-limb blood flow of the non-contracting, passive contralateral calf was measured by mercury in Silastic strain gauge plethysmography (Whitney, 1953; (AG101 Air Source, E20 Rapid cuff inflator, EC6 strain gauge plethysmograph, Hokanson, Bellevue, WA, USA). The strain gauge was positioned around the widest portion of the calf, and the leg supported in a dependent position at the foot and the knee to eliminate muscle tension. A cuff (CC17, Hokanson, Bellevue, WA, USA) was placed around the passive thigh and inflated to a pressure sufficient to prevent venous outflow. Venous occlusion pressure was calculated as 50 mmHg plus the sum of the distance from the centre of the thigh to the heart, multiplied by 0.779, accounting for the specific gravity of the blood (1.055; Gamble et al. 1997). The thigh cuff was rapidly inflated for 5 s every 10 s throughout the protocols. During the protocol, the blood flow was maintained to the foot (Siggaard-Anderson, 1970). Vascular conductance (in ml−1 min−1 (100 ml)−1 mmHg−1) was calculated by dividing blood flow by mean arterial blood pressure (MAP). In addition, respiration was measured using a Pneumotrace (model 1132, UFI, Moro Bay, CA, USA).
Post-ganglionic MSNA was measured from the non-active lower limb whilst the subject was seated (Vallbo et al. 1979; Wallin & Eckberg, 1982). Multi-unit recordings of MSNA were obtained with unipolar tungsten microelectrodes (Department of Clinical Neurophysiology, Karolinska Hospital, University of Stockholm, Sweden). The peroneal nerve was located using a probe, which discharged short-lasting (2 ms) stimulations (40–100 V) at 1 Hz (Grass-Telefactor, West Warick, RI, USA), to induce foot dorsiflexion and eversion. This was repeated using the recording needle at a much lower voltage (3 V) for precise location of the peroneal nerve. A reference needle electrode was also inserted near to the fibular head. The neural signals were amplified (95.5 × 103), filtered (bandwidth 700– 2000 Hz), rectified and integrated (time constant 0.1 s) to obtain a mean voltage neurogram (Nerve Traffic Analyser, model 662C-3, University of Iowa, Department of Bioengineering, Iowa City, IA, USA). A recording was considered acceptable when the neurogram revealed spontaneous pulse-synchronous bursts, with a minimum signal-to-noise ratio of 3:1 that increased during breath-holding manoeuvres. The absence of a response to arousal (loud noises) or skin stroking was used to discriminate between muscle and skin sympathetic nerve activity (Sundlof & Wallin, 1977). The recording was allowed to stabilize for at least 10 min before data were recorded for analysis. One researcher randomised the neurograms and another, who was blinded to the subject identity and experimental condition, performed scoring. MSNA was expressed as burst frequency (in bursts min−1), burst height and as total activity. Total activity was calculated as the product of average burst height and burst frequency, and is expressed in arbitrary units (a.u.).