Measurements

NIRS-derived deoxy[Hb+Mb] and total [hemoglobin + myoglobin] (total[Hb+Mb]) were measured continuously in the VL muscle of the right leg by means of NIRS (Oxiplex TS; ISS, Champaign, USA) at a sampling rate of 2 Hz throughout the entire protocol. The source detector differences were 2.0, 2.5, 3.0, 3.5 cm. Further details on the specifics of this system can be found elsewhere [3,26]. The NIRS probe was placed on the belly of the VL muscle midway between the inguinal crease and the proximal border of the patella and was secured in place by double-sided tape and an elastic strap to prevent any movement. The probe was covered by an optically dense, black vinyl sheet and an elastic bandage to minimize both the intrusion of external light and movement of the probe. A Harpenden skinfold caliper was used to measure skin and adipose tissue thickness at the area of the NIRS probe interrogation on the VL.

A breath-by-breath metabolic cart (Quark CPET, Cosmed, Rome, Italy) was used to measure gas-exchange variables and pulmonary ventilation (inspired and expired flow rates) starting at the beginning of the pre-RI test baseline at which point it was synchronized with the NIRS measurement. Expired gases were sampled at the mouth and analyzed for fractional concentrations of O₂ and CO₂ after calibration with precision-analyzed gas mixtures, according to manufacturer specifications. The flowmeter was calibrated using a syringe of known volume (3 liters).