ActiGraph acceleration data are processed through several steps (including band-pass filtering to attenuate unwanted signal frequencies) to generate the activity counts commonly used in physical activity research. We performed three experiments to investigate the effect of sampling frequency on the generation of activity counts. Ideal acceleration signals were produced in the MATLAB software. Thereafter, ActiGraph GT3X+ monitors were spun in a mechanical setup. Finally, 20 subjects performed walking and running wearing GT3X+ monitors. Acceleration data from all experiments were collected with different sampling frequencies, and activity counts were generated with the ActiLife software. With the default 30-Hz (or 60-Hz, 90-Hz) sampling frequency, the generation of activity counts was performed as intended with 50% attenuation of acceleration signals with a frequency of 2.5 Hz by the signal frequency band-pass filter. Frequencies above 5 Hz were eliminated totally. However, with other sampling frequencies, acceleration signals above 5 Hz escaped the band-pass filter to a varied degree and contributed to additional activity counts. Similar results were found for the spinning of the GT3X+ monitors, although the amount of activity counts generated was less, indicating that raw data stored in the GT3X+ monitor is processed. Between 600 and 1,600 more counts per minute were generated with the sampling frequencies 40 and 100 Hz compared with 30 Hz during running. Sampling frequency affects the processing of ActiGraph acceleration data to activity counts. Researchers need to be aware of this error when selecting sampling frequencies other than the default 30 Hz.