Background: Day-to-day walking variability appears not to be random and may fluctuate by day of week or type of day (workdays/non-workdays). The day-of-the-week variability is less clear in period of life transition between the ages of 50–70 years. Moreover, it is unclear whether the variability is related to age or BMI. Objective: The aim of this study was to identify the weekdays–weekends patterns and the day-to-day variability in step patterns in men and women of different age and BMI groups. Methods: During the years 2009–2012, 1,170 individuals (532 men and 638 women) aged 50–70 years participated in a seven-day monitoring using a Yamax Digiwalker SW-700 pedometer. Conclusion: In conclusion, this study contributes to the evidence with the findings about the existing the day-of-the-week variability of step counts per day in the population aged 50 to 64 years and about the absence of significant variability in seniors. Sunday was the least active day in different age and BMI groups aged 50 to 70 years of Czech population. Researchers and practitioners should take the results of this study into consideration when preparing for data collection of habitual physical activity in senior population and in the development of walking interventions and weight reduction programmes designed for this age group.