Percutaneous needle electrolysis (PNE) consists of the ultrasound-guided application of a galvanic electrical current through a solid filament needle. One proposed therapeutic mechanism for this intervention is a potential thermal effect. The aim of this study was to investigate if the application of PNE induces changes in temperature in different cadaveric musculoskeletal tissues. A repeated measure experimental cadaveric study was designed with 10 cryopreserved knees (5 men, 5 women). Sterile stainless-steel needles of 40 mm length and 0.30 mm caliber were used in this study. An ultrasound-guided needling puncture was performed in the targeted tissue (patellar tendon, infra-patellar fat, and vastus medialis muscle). Additionally, the tip of the needle was placed next to the thermometer sensor at the minimum possible distance without direct contact with it. The temperature differences before and after different applications were measured. The applications were: three applications for 3 s of 3 mA of intensity (3:3:3) when the tendon was the targeted tissue, three applications for 3 s of 1.5 mA of intensity (1.5:3:3) when the fat or muscle was the targeted tissue, and 24 s of 1 mA of intensity (1:24:1) in all tissues. No statistically significant Group*Time interactions were found in any tissue (tendon: F = 0.571, = 0.459, ŋ = 0.03; fat pad: F = 0.093; = 0.764, ŋ = 0.01; muscle: F = 0.681; = 0.420, ŋ = 0.04). Overall, no changes in temperature were observed between both applications in the tendon (3:3:3 vs. 1:24:1) and fat/muscle (1.5:3:3 vs. 1:24:1) tissues. The application of two different percutaneous needle electrolysis protocols did not produce appreciable thermal changes in the tendon, fat, and muscle tissues of human cadavers. The results from the current cadaver study support that a thermal effect should not be considered as a mechanism of clinical action regardless of the targeted human tissue when applying percutaneous needle electrolysis since no changes in temperature after its application were observed.