[34] and Malm et al. [35] was used to induce the eccentric muscle injury. After a 10-min warm-up at a speed chosen by the subject, subjects ran downhill (treadmill grade -10%) at a constant speed for 45 minutes. Running speed during the 45-min exercise was to be maintained at the anaerobic threshold, which was determined prior

to the test by measurement of lactate concentration in capillary blood during a 5-min run at a treadmill inclination Ro 61-8048 ic50 of 3%. A speed corresponding to a lactate concentration of 3.5-5 mmol/L was considered appropriate and therefore maintained throughout the exercise protocol. Subjects performed ten-min exercise bouts during the week prior to the study day (days -7 and -5) to familiarize with the exercise protocol and to break down more susceptible muscle fibres, in order to achieve similar

fibre composition and standardize the baseline level in all subject [36, 37]. One hour before the eccentric injury protocol all subjects received an oral nutritional supplement containing 25 to 30 g of carbohydrates and 2–4 g of protein. Also, hydration was assured by consumption of approximately 500 mL of mineral water from 30 min. prior to the start of the test. Subjects were allowed to drink water during the test. Magnetic resonance CX 5461 imaging (MRI) A high magnetic field system was used (Signa 1.5 T, G.E. Milwaukee, WI, USA). Images were acquired 48 hours after exercise, with the subjects in the supine decubitus position. Both thighs were explored. The diagnosis was based on MRI signal alterations in any muscular group both in the flexor and the extensor compartment, as well as on signal asymmetry as compared with the contralateral homonymous muscular group. The radiologist was blinded to the treatment group. Five non-contiguous axial

PRKD3 imaging slices (2-mm thickness, 2-mm gap) were selected. In order to quantify muscle injury, each thigh was divided into three compartments (anterior, posterior, medial) (Figure 1). A compartment was considered positive for muscular injury when an area of high signal intensity on T2-weighted and STIR sequences was observed in at least one muscle. Figure 1 STIR sectional image of both thighs in the middle third. Asterisk marks muscle area with increased uptake. Muscle biopsies Muscle biopsies were performed 48 hours after exercise to obtain samples for the analysis of markers of cellular injury (muscle myeloperoxidase [MPO] activity, immunohistochemical analysis of albumin [38] and CD3 positive cells). A skin incision was performed with a 5 mm blade. The same skin incision was used for both muscle biopsies, changing the needle direction [34, 39] . Two biopsies were carried out from the middle third of each vastus lateralis, under ultrasound control. Muscle samples were obtained using a Vacora System Biopsy gun (Bard Medical Systems, Tempe, AZ, USA), with a coaxial needle of 10G × 140 mm.