**3. Methodological and technical considerations**

The most used MUNE technique relays on manual incremental stimulation of the motor nerve, known as the McComas technique (McComas, 1995), modified by Ballantyne and Stålberg. The following test settings were used: sweep duration 50 ms, gain 2 mV/Div for M wave, 0.5 mV/Div for each step; filters 20 – 10 KHz (Keypoint Clinical Manual, 1999). The use of specific software for MUNE detects "alternation", eliminates subjectivity and the sampling of artifactually small motor units in ALS patients (McComas, 1995; Hong et al., 2007); ten incremental steps are commonly recorded (Sartucci et al., 2007).

Percutaneous stimuli were delivered over musculocutaneous nerve immediately below axilla, recording from BB muscles, and ulnar nerve at the wrist by recording from the ADM muscle of the same upper limb (Sartucci et al., 2007; Sartucci et al., 2011). Signals are detected with common surface electrodes, Ag/AgCl type, tapered on the cutis over the target muscles with a common muscle-belly tendon montage. In those patients who underwent follow-up after several months, each test was performed exactly on the same side with the same electrode position (spatial coordinates have been annotated in patients schedule).

At least two consecutive MUNE measures are usually performed on each patient to verify the consistency of our results; when required, further estimation was made until the MUNE was clearly stable. The mean of the two or more tests was calculated (Henderson et al., 2007). The results showed an excellent reproducibility with test-retest correlation coefficients ranging from 0.75 to 0.86 (Sartucci *et al.*, 2007).

The standard macro-EMG method is routinely applied in our patients (Stålberg, 1983). We employ a recording electrode, consisting of a modified single fibre EMG (SFEMG) electrode with the cannula Teflon insulated except for the distal 15 mm. The SFEMG recording surface is exposed 7.5 mm from the tip and the recording is made using two channels: the first one in whom the SFEMG activity is displayed (using the cannula as reference) and used to identify the MU and trigger the averaging procedure (band-pass filter for this channel: 500- 10 kHz); fiber density (FD) of the triggering single fibre electrode is recorded. The second channel averaged the activity from the cannula until a smooth baseline and a constant macro MUP was obtained (Filter pass-band: 5-10 kHz).

Total area between the curve and the baseline, the maximal peak-to-peak amplitude (macro-MUP) during the total sweep time of 70 ms are measured (Bauermeister and Jabre, 1992). Results are expressed as individual area values from at least 20 recordings. The relative macro amplitude is expressed as the obtained mean value (Stålberg, 1983). Fibre density is expressed as number of time locked spikes obtained on the SFEMG channel (Sanders and Stålberg, 1996).
