Background: Prolonged sedentary behaviour is associated with poor health outcomes. Office workers often engage in excessive sedentary behaviour, however limited research reports on how this sedentary behaviour is accumulated. This study examines objectively measured patterns of prolonged sedentary behaviour in female office workers during weekdays and weekend days and across time of day. Methods: Full time female office workers from a Scottish University participated (N = 27 mean age 43.0 ± 11.5 yrs; BMI 25.8 ± 4.1 kg/m2). Participants wore an activPAL™ for 7 days and completed a diary of waking and working hours. Average week and weekend time sitting, standing and stepping was calculated and also expressed as a proportion of waking day. Average week and weekend daily step count and sit to stand transitions were calculated. Continuous bouts of sedentary behaviour were categorised as: 20–40, 40–60 and > 60 minutes and compared between week and weekend days and across time of day. Results: Average weekday sitting time and proportion was higher (P < 0.05) than weekend days [9.1 hrs (66%) vs 8.1 hrs (56%)]. Time and proportion spent standing was higher (P < 0.01) at weekends than weekdays [4.6 hrs (32%) vs 3.8 hrs (27%)]. Time spent stepping [weekday 1.8 hrs (12%) vs weekend 1.7 hrs (12%)] and total daily step count (weekday 8903 vs weekend day 8014) were not significanlty different (P > 0.05) on weekdays vs weekend days. The pattern of sedentary behaviour bouts was different between week and weekend days. Week days were dominated by a consistent pattern of shorter (20–40 mins) sedentary behaviour bouts. The longest continuous sedentary behaviour bouts occurred in the evening, particularly at weekends. Conclusions: In office workers the most prolonged sedentary behaviour occurred in the evening, particularly at weekends. Interventions need to target these highly saturated periods of sedentary behaviour.