This study examined the optimal measurement conditions to obtain reliable peak cadence measures using the accelerometer-determined step data from the National Health and Nutrition Examination Survey 2005-2006. A total of 1282 adults (> 17 years) who provided valid accelerometer data for 7 consecutive days were included. The peak 1- and 30-minute cadences were extracted. The sources of variance in peak stepping cadences were estimated using Generalizability theory analysis. A simulation analysis was conducted to examine the effect of the inclusion of weekend days. The optimal number of monitoring days to achieve 80% reliability for peak stepping cadences were estimated. Intraindividual variability was the largest variance component of peak cadences for young and middle-aged adults aged < 60 years (50.55%-59.24%) compared with older adults aged ≥ 60 years (31.62%-41.72%). In general, the minimum of 7 and 5 days of monitoring were required for peak 1- and 30-minute cadences among young and middle-aged adults, respectively, whereas 3 days of monitoring was sufficient for older adults to achieve the desired reliability (0.80). The inclusion of weekend days in the monitoring frame may not be practically important. The findings could be applied in future research as the reference measurement conditions for peak cadences.