A functional evaluation of skeletal muscle oxidative metabolism during dynamic knee extension (KE) incremental exercises was carried out following a 35-day bed rest (BR) (Valdoltra 2008 BR campaign). Nine young male volunteers (age: 23.5 ± 2.2 yr; mean ± SD) were evaluated. Pulmonary gas exchange, heart rate and cardiac output (by impedance cardiography), skeletal muscle (vastus lateralis) fractional O 2 extraction, and brain (frontal cortex) oxygenation (by near-infrared spectroscopy) were determined during incremental KE. Values at exhaustion were considered “peak”. Peak heart rate (147 ± 18 beats/min before vs. 146 ± 17 beats/min after BR) and peak cardiac output (17.8 ± 3.3 l/min before vs. 16.1 ± 1.8 l/min after BR) were unaffected by BR. As expected, brain oxygenation did not decrease during KE. Peak O 2 uptake was lower after vs. before BR, both when expressed as liters per minute (0.99 ± 0.17 vs. 1.26 ± 0.27) and when normalized per unit of quadriceps muscle mass (46.5 ± 6.4 vs. 56.9 ± 11.0 ml˙min±1˙100 g -1). Skeletal muscle peak fractional O 2 extraction, expressed as a percentage of the maximal values obtained during a transient limb ischemia, was lower after (46.3 ± 12.1%) vs. before BR (66.5 ± 11.2%). After elimination, by the adopted exercise protocol, of constraints related to cardiovascular O 2 delivery, a decrease in peak O 2 uptake and muscle peak capacity of fractional O 2 extraction was found after 35 days of BR. These findings suggest a substantial impairment of oxidative function at the muscle level, “downstream” with respect to bulk blood flow to the exercising muscles, that is possibly at the level of blood flow distribution/O 2 utilization inside the muscle, peripheral O 2 diffusion, and intracellular oxidative metabolism. Copyright © 2011 the American Physiological Society.