<h3>Purpose/Objective(s)</h3> No standardized method objectively evaluates cancer pain. In this case, measuring heart rate variability (HRV) using fluctuations in heartbeat interval (statistical software) may be useful because pain activates sympathetic nerve activity. Fluctuation analysis of RRI frequency demonstrates a high frequency (HF) and low frequency (LF) component. HF represents parasympathetic nerve activity, while the LF-to-HF ratio (LF/HF) represents sympathetic nerve activity. We hypothesized that HRV measurement is an effective and objective evaluation tool for bone metastases pain. <h3>Materials/Methods</h3> This prospective study enrolled patients receiving radiotherapy for painful bone metastases. Pain was assessed using a numerical rating scale (NRS), and anxiety and depression were evaluated based on Hospital Anxiety and Depression Scale (HADS). The average, maximum, and minimum NRS scores were separately evaluated. Autonomic and physical activities were evaluated by measuring HRV through a wearable device (Union Tool Co., Tokyo, Japan). Through a three-dimensional conformal technique, radiotherapy was delivered as 20–30 Gy doses in 5–10 fractions. Alternatively, stereotactic body radiotherapy (SBRT) was delivered as doses of 24 Gy in two fractions or 35 Gy in five fractions. In principle, the values of NRS, HADS, and RRI were obtained upon starting, completing, and 3–5 weeks after radiotherapy. RRI were obtained over approximately 24 h. <h3>Results</h3> Between July 2020 and July 2021, 11 patients were enrolled. The median age was 65 years (range: 50–83 years). Seven of 11 patients were treated with SBRT. We analyzed 27 NRS scores and heart rate interval measurements and 26 HADS scores. The median, average, maximum, and minimum NRS scores were 5 (range: 2–10), 6 (range: 2–10), and 3 (range: 0–8), respectively. HADS-assessed median anxiety and depression scores were both 8 (ranges: 1–13 and 2–21 respectively), and mean HF and LF/HF values were 0.52 ms<sup>2</sup> (SD: 1.66 ms<sup>2</sup>) and 2.45 (SD: 1.20), respectively. For all samples, NRS score had no significant relationship with mean LF/HF or HF (p = 0.66 and p = 0.74, respectively). However, for samples with NRS scores ≥ 4, NRS score had a significant association with LF/HF (p = 0.03). Heart rate during physical activity was significantly higher than the resting heart rate; however, mean resting LF/HF was significantly higher than LF/HF during physical activity. During rest, mean LF/HF was not associated with NRS score across all samples, including those with NRS scores ≥ 4 (p = 0.61 and p = 0.49, respectively). In samples excluding those with NRS scores < 4 and HADS depression scores ≥ 7, NRS score had a weak significant relationship with mean LF/HF (p = 0.07). <h3>Conclusion</h3> HRV measurements could objectively evaluate bone metastases pain. However, the effects of mental status, such as depression, on LF/HF affects HRV in cancer patients with mild pain.