Because of their extended fasting period on land during breeding, male king penguins have been extensively studied in order to unravel the physiological adaptations that enable them to fast while having to find a partner, defend their territory, or brood their offspring. While the different phases of fasting and the nature of the metabolic fuels used are well characterized in male king penguins, few studies have focused on the efficiency of the conversion of the metabolic resources into energy at a cellular level through mitochondrial respiration. Furthermore, little information is available on females in general while they experience fasting periods. Here, we measured mitochondrial respiration rates of red blood cells (RBCs) at the beginning (3 d) and end (10 d) of a natural egg-incubation fast in male and female king penguins. We tested whether RBC mitochondrial metabolism and its efficiency are modulated by fasting duration in free-living king penguins but also assessed whether this modulation is sex specific. In response to fasting, the respiration allocated to ATP synthesis in RBCs decreased in both sexes. Interestingly, RBC mitochondrial metabolic rates were higher in females at any stage of fasting. Furthermore, RBC mitochondrial metabolism efficiency decreased in males after 10 d of fasting but remained constant in females. Our results demonstrate that RBC mitochondrial metabolism is context and state dependent, differing between sexes and changing with fasting. They underline the importance of taking both sexes into account in physiological studies, where females remain underrepresented.