Abstract The short‐term response of humpback whales to boat approach and remote biopsy sampling was investigated in a breeding ground according to age‐class, sex, female reproductive status, social context, sampling system, habitat, and repeated sampling with more than 20 years of data. In a total of 2,248 observed behavioral responses to biopsy sampling, 58.7% showed no response, 40.7% were considered to be weak or moderate responses, and only 0.5% were considered to be strong. No significant difference was found between the response of calves (young‐of‐the‐year) and that of adults, whereas juveniles responded significantly more strenuously. The response of adult whales was influenced by sex, female reproductive status, and social context. Mothers showed the most frequent response to the boat approach, but the weakest response to the biopsy sampling itself, whereas nonlactating females had the strongest response to biopsy sampling. Males responded less when sampled within a competitive group. Repeated sampling of individual adults resulted in decreasing intensity of responses across years. This comprehensive assessment has implications for management authorities, notably in approving research permits for sampling different age classes on breeding grounds.