Physical activity is associated with health. The aim of this study was (a) to access if Portuguese university students meet the public health recommendations for physical activity and (b) the effect of gender and day of the week on daily PA levels of university students. This observational cross-sectional study involved 126 (73 women) healthy Portuguese university students aged 18-23 years old. Participants wore the ActiGraph wGT3X-BT accelerometer for seven consecutive days. Number of steps, time spent sedentary and in light, moderate and vigorous physical activity were recorded. The two-way MANOVA revealed that gender (p-value = 0.001; η2 = 0.038; minimum effect) and day of the week (p-value = 0.001; η2 = 0.174; minimum effect) had significant main effects on the physical activity variables. It was shown that during weekdays, male students walked more steps (65.14%), spent less time sedentary (6.77%) and in light activities (3.11%) and spent more time in moderate (136.67%) and vigorous activity (171.29%) in comparison with weekend days (p < 0.05). The descriptive analysis revealed that female students walked more steps (51.18%) and spent more time in moderate (125.70%) and vigorous (124.16%) activities during weekdays than in weekend days (p < 0.05). Women students did not achieve the recommended 10,000 steps/day on average during weekdays and weekend days. Only male students achieved this recommendation during weekdays. In summary, this study showed a high incidence of sedentary time in university students, mainly on weekend days. New strategies must be adopted to promote physical activity in this population, focusing on the change of sedentary behaviour.