This study investigated the effects of night work on performance efficiency. Night work is generally acknowledged to impair performance, yet much research has contradicted this assertion. The feasibility of including brief periods of physical activity to stimulate arousal within mentally demanding work shifts was also evaluated. Thirty six postgraduate volunteers were assigned to either the cognitive tasks (CT) or cognitive and motor tasks (CMT) group. All subjects performed three psycho-motor tests, using the Vienna Test System, at midday and midnight. The CMT group performed a short cycling activity before each test. Heart rate responses served as physiological measures, the Perceived Strain Scale was used to quantify individual perceptions of strain and performance efficiency was assessed in terms of speed and accuracy. Although several trends were apparent, no significant differences (p < 0.05) were revealed with respect to the three performance variables between the midday and midnight test sessions, or between the CT and CMT subjects, other than the higher heart rates recorded in the CMT group. In summary, neither time of day nor physical activity were found to affect performance within the controlled environment of this study.