Myasthenia gravis (MG) is the autoimmune disorder affecting the neuromuscular junction with morbidity estimated at 70-320 per 1 000 000. The main symptom is the fatigue, which increases gradually through the day and also after exercise. The repetitive nerve stimulation (RNS) remains the key diagnostic test. In RNS, the peripheral nerve is electrically stimulated with trains of repetitive pulses at constant (e.g. 3Hz) frequency. The electrical responses associated with contraction of the one of respectively innervated muscles are recorded. While in healthy subject the amplitudes of subsequent responses should be similar, in MG they gradually decrease reflecting the insufficiency of neuromuscular junction and being the electrophysiological correlate of the fatigue. RNS is relatively simple, safe as well as time and cost-effective procedure. However, its sensitivity is not satisfactory. Even when voluntary exercise and other provocative methods are implemented, the sensitivity does not exceed 89% in generalized form of MG (where symptoms affect bulbar, limb and other muscles) and 70% in ocular form (where symptoms are limited to bulbar muscles). In some laboratories the sensitivity of detecting the impaired neuromuscular transmission can be increased with the use of the single fiber electromyography. This method is however not widely available as it requires skilled personnel and dedicated software. It is also not specific for MG. Therefore, research effort is still being done to increase the sensitivity of the RNS. The well-known gradual exacerbation of MG-related fatigue through the day let us to hypothesize, the RNS performed in the evening may be more sensitive than in the morning. The primary objective of this study will be therefore a comparison of RNS sensitivity performed in these both times of the day. Additionally, investigators want to assess if adding the second voluntary effort could be another way of improving the RNS-sensitivity. The maximal voluntary effort of the investigated muscle, lasting between 30 seconds and one minute is typically done in the course of the RNS. Usually, one or several trains of five to ten impulses precede it and also several trains follow it. While prolonging the effort over one minute may cause discomfort or problems with adherence, adding second one-minute effort, separated from the first by several trains should be suitable and well tolerable. It will prolong the whole procedure by approximately six minutes minutes.

Diagnostic procedure: In every recruited patient, RNS will be done thrice at 8:30 a.m., 8:50 a.m. and 2:30 p.m. on the same day or at 8:30 a.m. and 8:50 a.m. on one day and at 2:30 p.m. on the subsequent day. To exclude peripheral neuropathy, before the first RNS, the sensory nerve conduction study will be performed on the sural nerve and the motor conduction study on the tibial nerve on the nondominant side. To exclude myopathy, before the first RNS, the needle electromyography will be performed on the biceps brachii, of dominant side. Also, before the first RNS, the Myasthenia Gravis - Activities of Daily Living Score (MG-ADL) will be filled out. Every RNS will comprise investigation of the nasalis and trapezius muscles on the clinically more affected side or on the nondominant side when lateralization will not be evident. Investigation of every muscle will include two, 3Hz trains of ten supramaximal electric stimuli, separated by 1 minute interval. Then the 1 minute of maximal voluntary effort will follow and after that six 3Hz trains of ten supramaximal stimuli, separated by 1-minute intervals. The first or the second RNS (at 8:30 or at 8:50 a.m.) will include additional 1 minute of maximal voluntary effort, followed by additional six 3Hz trains of ten supramaximal stimuli, separated by 1-minute intervals. The allocation of the additional voluntary effort and subsequent six trains to the first or to the second RNS will be done randomly. The randomization list will be created via the website www.randomization.com using blocks of five subjects. The amplitude drop (the decrement) of the fourth response (out of 10 in every train) \>=10% with respect to the first response will be considered abnormal (indicative of impaired neuromuscular transmission). The whole RNS will be considered abnormal (indicative of impaired neuromuscular transmission) when the decrement \>=10% will be repeatable in at least several trains in at least one out of two tested muscles. The temperature of the skin will be maintained at 35 Celsius. If being taken, the acetylcholinesterase inhibitors will be withheld 12 hours before the first RNS.