PURPOSE藧: The current study aimed at assessing the effect of a trial of two nights of sleep deprivation (SDT) on mood, sleepiness, motivation and cognitive and motor performance. METHODS藧: Thirty-six healthy young and physically active adult men (17 in the control group and 19 in the SDT group) completed a 48-h control or 48-h SDT. For the SDT, participants did not sleep for 48 h. Executive function (attention and inhibitory control) in the Go/No-Go and Stroop tests, mood, sleepiness, motivation, heart rate variability (HRV), motor performance in a hand grip strength test, and 60-s maximal isometric contraction (MVC-60 s) of knee extension were evaluated at 9-11 am on consecutive days 1, 2, and 3. RESULTS: One night of sleep deprivation increased sleepiness, decreased mood, motivation and motor endurance but did not affect executive function (as measured in the Stroop and Go/No-Go tests), the MVC for hand and leg knee extensor muscles, and peripheral motor fatigue in the leg MVC-60 s task. However, the central activation ratio (CAR) decreased significantly during the MVC-60 s. The SDT significantly contributed to the decrease in these functions. That is, the SDT reduced executive function (increased reaction time during Go/No-Go test), MVC of knee extension, and the CAR before and after the MVC-60 s. By contrast, the SDT did not increase CAR immediately after the MVC-60 s and did not decrease the rate of torque development (RTD). CONCLUSIONS藧: The SDT significantly impaired mood, motivation and increased sleepiness and HRV, reduced MVC of knee extensor muscles (but not RTD) and motor performance during the MVC-60 s and worsened executive function (attention and inhibitory control) only during the Go/No-Go task. However, the SDT did not reduce hand grip strength and CAR immediately after the MVC-60 s of knee extensor muscles.