Algometers are commonly used to measure the pain-pressure threshold (PPT) in various tissues, such as muscle, tendons, or fascia. However, to date, it is not clear if the repeated application of a PPT assessment can adjust the pain thresholds of the various muscles. Therefore, the purpose of this study was to investigate the repeated application of PPT tests (20 times) in the elbow flexor, knee extensor, and ankle plantar flexor muscles in both sexes. In total, 30 volunteers (15 females, 15 males) were tested for their PPT using an algometer on the respective muscles in random order. We found no significant difference in the PPT between the sexes. Moreover, there was an increase in the PPT in the elbow flexors and knee extensors, starting with the eighth and ninth assessments (out of 20), respectively, compared to the second assessment. Additionally, there was a tendency to change between the first assessment and all the other assessments. In addition, there was no clinically relevant change for the ankle plantar flexor muscles. Consequently, we can recommend that between two and a maximum of seven PPT assessments should be applied so as not to overestimate the PPT. This is important information for further studies, as well as for clinical applications.