Energy conservation has long been a focal point in hibernation research. A long-standing assumption is that ambient temperature (Ta) largely defines the rate of energy expenditure because of well-known relationships between Ta, metabolic rate and frequency of arousal from torpor. Body condition and humidity also affect energy expenditure but are usually considered secondary factors. We held tricolored bats (Perimyotis subflavus) in captivity under multiple environmental conditions to directly compare the importance of Ta, fat mass and humidity for hibernation energy expenditure. Fat mass was the best predictor of female mass loss, followed by Ta and humidity. However, males had less fat and adopted a more energetically conservative hibernation strategy. Our results demonstrate that understanding the evolution of behavior, physiology and ecology of hibernation requires disentangling the relative contributions of multiple drivers of hibernation energetics, and that Ta is not always the most important factor driving energy expenditure.

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