BACKGROUND: Many family caregivers of patients with cancer feel guilty about self-care. A meaningful relationship with patients reduces such negative feelings and functions as self-care for family caregivers. Moreover, handholding improves autonomic functions in non-cancer patients. However, the effects of handholding on both patients with cancer and family caregivers remain unknown. METHODS: We evaluated the effects of handholding on heart rate variability (HRV) in patients with cancer and their family caregivers. This randomized crossover study divided patients with cancer and their family caregivers into two trial groups: Handholding trial (the family caregiver holds the patient\'s hand for five minutes) and Beside trial (the family caregiver stays beside the patient without holding their hand). The study included 37 pairs of patients with cancer who received treatment in the cancer department of a university hospital in Japan and their family caregivers (n = 74). The primary end-point was the change in HRV before and during the intervention. RESULTS: The median performance status of the patients was 3. An interaction was observed between trials in the standard deviation of the normal-to-normal interval (SDNN) of HRV for family caregivers (F = 7.669; p = 0.006), and a significant difference in time course was observed between the trials (before p = 0.351; during p = 0.003). No interaction was observed between trials in the SDNN for patients (F = 0.331; p = 0.566). Only a main effect in time course (F = 6.254; p = 0.014) was observed. SDNN increased significantly during the intervention in both trials (Handholding trial: p = 0.002, Beside trial: p = 0.049). CONCLUSIONS: Handholding improves autonomic functions of family caregivers and may function as self-care for family caregivers. TRIAL REGISTRATION: UMIN000020557 . Registered on January 15, 2016.