The confinement and lockdown imposed by the COVID-19 pandemic have produced restrictions in the lifestyle of Italian citizens with variations in their psychological well-being. The aim of the study was to identify changes and relationship with socio-demographic parameters. An online survey was administered to 1383 subjects (1007 females and 307 males) working in the University of Florence, Italy. Three validated questionnaires were used for the survey: the Global Physical Activity Questionnaire, the Med Diet Score and the Psychological General Well-Being Index-A. All the subjects were asked to complete the questionnaires twice, in order to attain a picture of the habits before and a later time point during confinement. Our results show that work-related physical activity was decreased, along with an increase in sedentary behaviour (from 07:22±03:20 to 08:49±03:41 h:min; p<0.001, ES = 0.38), whereas recreational physical activity was increased (vigorous exercise varied from 568.5 ± 838.6 to 833.7 ± 1263.0 METs; p<0.002, ES = 0.25). Eating habits changed according to the place where meals were eaten, with an increased habit for breakfast and snacks and a slight increase in alcohol consumption. Psychological well-being decreased (Index from 21.4±3.9 to 18.0±5.3; p<0.001, ES = 0.723), especially in terms of vitality and positive thinking. The socio-demographic variables affecting these variations were mostly represented by age, gender and working conditions: young age and self-employment conditions can be considered factors for the changes in daily habits induced by confinement that may affect psychological well-being.