# Effect of PEMF on Muscle Oxygen during Cycling:
## 2 Materials and Methods
### Experimental Procedures  
Each participant visited our laboratory 5 times over a 4-wk period, during which we performed different recordings. During visit 1, we recorded an incremental test to exhaustion on a cycle-ergometer (H-300-R Lode), necessary to individualize the workload for the succeeding recording sessions through the detection of ventilatory threshold (VT) and peak oxygen consumption (VO2 peak). Participants performed 5 min of warm-up cycling at 50 W, after which the work rate, starting at 80 W, was increased by 20 W/min until the volitional exhaustion [17; 18]. Participants cycled at 70-80 rpm and this pedal rate was reproduced in subsequent constant-load exercises. Breath by breath pulmonary gas exchange values were recorded non-stop during the incremental exercise tests and averaged every 1 s. The VO2 peak was taken as the highest 20 s mean value attained until volitional exhaustion. VT was obtained considering: (i) the first unbalanced rise in CO\({}_{2}\) production (VCO\({}_{2}\)) from visual examination of individual plots of VCO\({}_{2}\) vs. VO\({}_{2}\); (ii) an increase in expired ventilation (V\({}_{\rm E}\)/VO\({}_{2}\)) with no increase in V\({}_{\rm E}\)/VCO\({}_{2}\); and (iii) an increase in end-tidal O\({}_{2}\) tension with no fall in end-tidal CO\({}_{2}\) tension. The data recorded during the incremental test were used to calculate the work rates used during the subsequent constant-load exercise tests. Precisely, the workload for each athlete (mean \(\pm\) SD: 308.2 \(\pm\) 61.8 watt) was set to ~50% of the difference between power reached at ventilatory threshold (VT) and at VO\({}_{\rm 2 peak}\) (~50% \(\Delta\) VT-VO\({}_{\rm 2 peak}\)). Expired gas was recorded using the Quark b2 breath-by-breath metabolic system ([PERSON], Rome, Italy). During the following four visits, athletes performed 6 min of heavy constant-load exercise both during active (ON) and inactive (OFF) PEMF stimulation. The four sessions were performed in random order. Two PEMF loop-antenna devices (Torino II, Rio Grande Neurosciences, USA) were positioned on the right leg, one at the upper and one at the lower thigh level. The PEMF waveform consisted of a pulse-burst modulated 27.12 MHz sinusoidal carrier, with a 2 ms burst width repeated at 2 Hz, with peak magnetic field at the center of the loop 5 \(\pm\) 1 \(\mu\)T. The two circular 20 cm loops were applied on the thigh in both experimental conditions (ON and OFF) because the stimulation was not perceived by the subjects at the cutaneous level (single-blind). Heavy constant-load exercise was performed on the same cycle-ergometer, under a standardized procedure, in a quiet chamber with a steady and comfortable temperature (22 \({}^{\circ}\)C), at the similar day time (9:00-12:00 AM) to prevent circadian effect [19; 20]. Subjects were asked to refrain from caffeine intake before the tests and were recommended to avoid physical activity and alcohol in the 12h prior the test. After 10 min of free warm-up and 5 min of rest, the athletes started the recording session with 1 min of unloaded cycling (baseline), followed by an instantaneous increase in the individualized power, which was attained in ~3s. Pulmonary O\({}_{2}\) uptake was collected with the Quark b\({}^{2}\) breath by breath metabolic system (Cosmed, Rome, Italy); meanwhile, for muscle oxygenation we used a near-infrared spectroscopy (NIRS, NIMO, Nirox s.r.l. Italy), a noninvasive and validated method which used different light absorptions of hemoglobin (Hb) and myoglobin related to their oxygen saturation [21]. To record HHb and TOI values during cycling, the NIRS probe, with a continuous wave with a sampling rate set at 40 Hz, with an emitter and detector pair, was firmly located on the skin above the lower third of the right vastus lateralis (VL) muscle (about 12 cm upon the proximal border of patella and 3 cm lateral to the midline of the thigh), and held with a band of Velcro straps [22]. Elastic strappings were placed around the muscle probe to avoid corruption from localized light.