Background The Chinese hospital ship “Peace Ark” has conducted nine overseas humanitarian missions. Methods Research indicated long-distance voyages significantly impact seafarers’ physical and mental health. This study investigated changes in and influencing factors for medical professionals’ health-related quality of life (HRQoL) during a 2017 humanitarian mission (8 countries, 155 days). Using a cohort study design, 90 medical professionals’ HRQoL was assessed (via the SF-12) pre-voyage and after 5 months at sea. Results Pre-voyage, mean physical component scale (PCS) and mental component scale (MCS) scores were 52.34 (SD 5.07) and 53.03 (SD 6.40), respectively—significantly higher than in the general Chinese population (p < 0.0001). Afterwards, PCS and MCS scores declined by 3.9867 (SD 6.03) and 5.70 (SD 7.03), respectively. PCS scores were negatively associated with female gender (p = 0.004). Compared to administrative staff, doctors (p < 0.001) and nurses (p < 0.001) showed less deviation in PCS scores. Lower MSC scores were associated with older age (p = 0.011) and less family support (p = 0.0165). Conclusions Long-term voyages negatively impacted HRQoL in medical personnel. Thus, prior to similar future humanitarian missions, selection criteria and training procedures for medical personnel should be optimized, thereby increasing their confidence and social support.