2.4. Exercise Tests
Moderate intensity constant-work rate cycling exercise. An electromagnetically-braked cycle ergometer (Corival; Lode, Groningen, The Netherlands) was utilized. Subjects exercised at their freely chosen pedal frequency (80 ± 5 rpm). Each subject performed two repetitions of a 6-min constant work rate moderate-intensity exercise (CWR). Transitions from unloaded pedaling to the imposed work rate were attained in ~3 s. The work rate was chosen to correspond to 50% of peak work rate reached during the incremental test.
Isometric knee extensions. Subjects were seated on a special chair, secured by a safety belt tightened around the shoulders and abdomen, with the arms grasping handlebars and the legs hanging vertically down. A strap was tightened around the subject’s dominant ankle, and was linked by a steel chain to a fixed frame. The chain length was regulated to obtain a knee angle of 110 degrees. The fixed frame was positioned behind the ankle to perform the isometric knee extensions. Subjects began the experimental session by performing a warm-up, which consisted of 20 sub-maximal isometric contractions at a self-selected intensity. After that, they performed two exercises in the same experimental session with the dominant lower limb only:
(A) Maximal voluntary contraction (MVC): subjects were asked to perform three MVCs of three-to-four seconds in duration each. To prevent fatigue, after each contraction subjects rested for two minutes. The highest force was multiplied by the moment arm in order to calculate maximal voluntary torque (MVT).
(B) Fatiguing intermittent submaximal knee extension: based on pilot studies, subjects performed intermittent isometric knee extensions of 3.5 s, with 10 s of rest between them. The target torque to reach and maintain during each contraction was set at 75% of the actual MVT (i.e., at the same relative intensity, in HND and CD). Two different auditory feedbacks were given to subjects: (1) a “ring”, preceded by a countdown, determined the start and the end of each contraction; (2) a monotonic sound highlighted the reaching of the torque target level. Experimental sessions ended when subjects were not able to reach the target torque for two consecutive contractions.
Repeated Sprint Ability test (RSA)
. RSA consisted of five “all out” 6-second sprints on a cycle ergometer (894E, Monark Exercise AB, Vansbro, Sweden) separated by 24 s of inactive recovery [32
]. Subjects pedaled in a seated position, and the mechanical resistance (F) was set at 0.74 N∙kg−1
Physiological variables. Pulmonary ventilation (), , and carbon dioxide output () were determined breath-by-breath by a computerized metabolic cart (Vmax29c; SensorMedics, Bilthoven, The Netherlands). Heart rate (HR) was determined from the electrocardiogram signal. Gain values (G)—the variable estimating the O2 cost of cycling—were calculated as Δ ( at the end of CWR minus resting ) divided by work rate. Blood lactate concentration ([La]b) was measured at rest and at several times during recovery on 20 µL of capillary blood obtained from a pre-heated earlobe by an enzymatic method (Biosen C-line; EKF Diagnostics GmbH, Barleben, Germany). The highest [La]b was taken as [La]b peak.
Force recording. A force sensor (TSD121C, BIOPAC Systems, Inc., Goleta, CA, USA) was connected in series to the chain, which connected the fixed frame of the special chair to the strap tightened around the subject’s right ankle. Force analog output was sampled at a frequency of 1 kHz using a data acquisition system (MP100, BIOPAC Systems, Inc., Goleta, CA, USA) connected to a personal computer by means of an USB port.
Surface Electromyography (EMG) recording
. EMG data were collected from the right (dominant) thigh: vastus lateralis (VL) was selected as the main knee extensor muscle. Pre-gelled surface EMG electrodes (circular contact area of 1 cm diameter, BIOPAC Systems, Inc., Goleta, CA, USA) were placed (inter-electrode distance equal to 20 mm) at two-thirds on the line from the anterior spina iliaca superior to the lateral side of the patella [33
]. In order to ensure a good electrode–skin interface, prior to the application of the electrodes, the subject’s skin was shaved, rubbed with an abrasive paste, and cleaned with a paper towel. EMG electrodes were placed at the beginning of the experimental session, and were not removed between the two exercises. The locations of the electrodes during the first experimental session were marked on the skin with a permanent ink pen. In order to place electrodes in the same positions prior to the second session, the subjects were asked to refresh these contours daily. To record the EMG data, the electromyography system (EMG100C, BIOPAC Systems, Inc., Goleta, CA, USA; Low Pass Filter: 500 Hz; High Pass Filter: 10 Hz; Noise Voltage (10–500 Hz): 0.2 µV (rms); Zin: 2 M ohm; CMRR: 110 dB) was used. EMG data were sampled at a frequency of 1 kHz using a data acquisition system (MP100, BIOPAC Systems, Inc., Goleta, CA, USA), and processed using the program LabChart 7 Reader (ADInstruments Pty Ltd., Bella Vista, NSW, Australia).
Force and EMG analysis
. As for MVC, a 500 ms window was centered at the maximal force exertion to calculate MVT (see above) and to analyze the surface electromyography (sEMG), its intensity being quantified by root mean square (RMS). During intermittent submaximal isometric contractions, no mechanical work is performed, so the torque-time integral (TTI) was used to estimate muscle work [34
]. For each single knee extension, TTI and RMS of vastus lateralis (RMS-VL)—expressed as a percentage of maximal voluntary contraction (%MVC)—were calculated. In addition, the average value of RMS-VL calculated over the first three knee extensions was compared to the one obtained during the last three knee extensions, in order to investigate the fatigue effect on muscle activation (adapted from Mulder et al., 2007) [35
Power recording. The power (P) values were calculated as P = F × d × RPM, where F is the resistance set (0.74 N∙kg−1 body mass), d is the distance covered by the flywheel at each revolution, and RPM is the number of revolutions per minute. Instantaneous P values were sampled at 50 Hz and then averaged each second. Peak Power (PP) was considered the maximal value of power recorded over a second.
Blood sampling. Resting blood samples were collected to determine plasma levels of nitrate and nitrite before the experimental phase and on day 6 of both diet periods, at least 2.5 h after the last meal. Venous blood was drawn from the antecubital vein into a 5-mL EDTA Vacutainer tube (Vacutainer, Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Plasma was immediately separated by centrifuge (5702R, Eppendorf, Hamburg, Germany) at 1000× g for 10 min at 4 °C. Plasma samples were then ultrafiltered through a 10 kDa molecular weight cut-off filter (AmiconUltra; Millipore, EMD Millipore Corporation, Billerica, MA, USA) using a ultracentrifuge (4237R, ALC, Milan, Italy) at 14,000× g for 60 min at 4 °C to reduce background absorbance due to the presence of hemoglobin. The ultrafiltered material was recovered and used to measure nitrite and nitrate concentration by the Griess method using a commercial kit (Cayman, BertinPharma, Montigny le Bretonneux, France). Samples were read by the addition of Griess reagents at 545 nm by a microplate reader spectrophotometer (Infinite M200, Tecan Group Ltd., Männedorf, Switzerland). A linear calibration curve was computed from pure nitrite and nitrate standard. All samples were determined in duplicate, and the inter-assay coefficient of variation was in the range indicated by the manufacturer.