Background: Retirement is a major life transition during which people restructure everyday activities; however little is known about this. The primary aim of the Life After Work study is to comprehensively measure changes in time use and patterns of physical activity and sedentary behaviour, and its associations with health and wellbeing, across the retirement transition. Methods/Design. A target sample of 120 participants aged 50 years and over will be recruited in two Australian state capital cities, Adelaide and Brisbane. Participants will undertake a battery of assessments approximately 3 months prior to retirement, and 3, 6 and 12 months post-retirement. Measures will include self-reported use of time (using the Multimedia Activity Recall for Children and Adults), objectively assessed physical activity and sedentary behaviour (using Actigraph GT3X+ accelerometers), self-reported health and well-being (using a battery of questionnaires including the Short-Form Health Survey (SF-36), Australian Unity Personal Well-being Index (AUPWI), Depression Anxiety Stress Scales 21 (DASS21), Short Warwick-Edinburgh Mental Well-being Scale, UCLA Loneliness Scale, Rosenberg Self-Esteem Scale), retirement circumstances and socio-demographic characteristics, objectively assessed anthropometric measures (height, weight and waist circumference), and resting blood pressure. Multivariate mixed models will be used to examine changes in use of time, health and well-being across retirement. Discussion. The results will provide important new information that will inform the development of lifestyle and policy interventions to address and improve health and well-being in retirement.