The purpose of the study is to determine the impact of local heating on skeletal muscle transcriptional response related to mitochondrial biogenesis and mitophagy. Twelve healthy subjects (height, 176.0 ± 11.9 cm; weight, 83.6 ± 18.3 kg; and body composition, 19.0 ± 7.7% body fat) rested in a semi-reclined position for 4 h with a heated thermal wrap (HOT) around one thigh and a wrap without temperature regulation (CON) around the other (randomized). Skin temperature, blood flow, intramuscular temperature, and a skeletal muscle biopsy from the vastus lateralis were obtained after the 4 h intervention. Skin temperature via infrared thermometer and thermal camera was higher after HOT (37.3 ± 0.7 and 36.7 ± 1.0 °C, respectively) than CON (34.8 ± 0.7, 35.2 ± 0.8 °C, respectively, p < 0.001). Intramuscular temperature was higher in HOT (36.3 ± 0.4 °C) than CON (35.2 ± 0.8 °C, p < 0.001). Femoral artery blood flow was higher in HOT (304.5 ± 12.5 mL‧min-1) than CON (272.3 ± 14.3 mL‧min-1, p = 0.003). Mean femoral shear rate was higher in HOT (455.8 ± 25.1 s-1) than CON (405.2 ± 15.8 s-1, p = 0.019). However, there were no differences in any of the investigated genes related to mitochondrial biogenesis (PGC-1α, NRF1, GAPBA, ERRα, TFAM, VEGF) or mitophagy (PINK-1, PARK-2, BNIP-3, BNIP-3L) in response to heat (p > 0.05). These data indicate that heat application alone does not impact the transcriptional response related to mitochondrial homeostasis, suggesting that other factors, in combination with skeletal muscle temperature, are involved with previous observations of altered exercise induced gene expression with heat.