Sample entropy (SaEn) has been used to assess the regularity of lower limb joint angles during walking. However, changing sampling frequency and the number of included strides can potentially affect the outcome. The present study investigated the effect of sample frequency and the number of included strides on the calculations of SaEn in joint angle signals recorded during treadmill walking. Eleven subjects walked at their preferred walking speed for 10 min, and SaEn was calculated on sagittal plane hip, knee, and ankle angle signals extracted from 50, 100, 200, 300, and 400 strides at sampling frequencies of 60, 120, 240, and 480 Hz. Increase in sampling frequency decreased the SaEn significantly for the three joints. The number of included strides had no effect on the SaEn calculated on the hip joint angle and only limited effect on the SaEn calculated on the knee and ankle joint signals. The present study suggests that the number of data points within each stride to a greater extent determines the size of the SaEn compared to the number of strides and emphasizes the use of a fixed number of data points within each stride when applying SaEn to lower limb joint angles during walking.