This pilot study is taking part into an experimental long run project which aims at improving road safety conditions for motorcyclists. Since car drivers’ performances are known to be sensitive to the effects of time of day and sleep deprivation, the influence of these two disruptive factors on two-wheelers riding capabilities remain to be identified. Eight subjects (motorcycle instructors) took riding tests at 06:00 h and 18:00 h either after a normal night’s sleep and after a night of total sleep deprivation. Our results revealed a significant improvement in riding performance between the sessions organized at 06:00 h and 18:00 h after a normal night’s sleep. However, in contrast with what has been observed in the case of car driving, this diurnal fluctuation did not occur after the night of total sleep deprivation. Such results may be caused by a significant deterioration in the motorcyclists’ riding performances during the 18:00 h session following the night of sleep deprivation. Consequently, it seems that the mechanisms implied are not the same as in automobile driving. Thus, it would be useful now, to beneficiate of the use of a motorcycle simulator to test precisely which are the resources implied in motorcycling and how they evolve.