Ultrasonic attenuation in calcaneus has been shown to be a useful measurement for the diagnosis of osteoporosis. Several studies indicate that this measurement is affected by temperature fluctuations, although the fundamental causes for this are currently not well understood. To investigate this phenomenon, six defatted human calcanei were interrogated in vitro at six temperatures ranging from 10°C to 40°C. The temperature-related variation was −0.18 dB/cmMHz°C (95% confidence interval: −0.27 dB/cmMHz°C, −0.10 dB/cmMHz°C). This study reinforces the notion, advanced by other investigators, that temperature-related effects need to be taken into account when performing diagnostic measurements that require high precision (such as monitoring responses to drug intervention), will aid in the interpretation of in vivo experiments designed to investigate temperature-dependent precision limitations, will facilitate comparisons between in vitro studies normally carried out at room temperature with in vivo studies carried out at body temperature, and fills a gap in the compendium of measurements of temperature-dependences of acoustic properties of biologic tissues.