Mice have become more important as genetically-modified model animals for analysis of physiological functions. The establishment of a frequent blood sampling system in conscious mice would provide a powerful tool for a better and more detailed understanding of the physiological status of circulating hormonal changes, such as pulse or surge modes of luteinizing hormone (LH) secretion. Frequent blood sampling, however, is considered problematic in mice because of the limited blood volume for their small body size. The present study, therefore, aims to establish a blood sampling protocol to determine the pulse and surge modes of LH secretion using intra-atrial cannulation and frequent blood sampling in free-moving conscious mice. Ovariectomized mice were bled every 3 min for 1.5 h to detect LH pulses. Blood glucose levels, an indicator of stress, were kept constant throughout the 1.5-h sampling period, suggesting that sampling can be performed under stress-free conditions. Obvious LH pulses were observed in ad lib-fed ovariectomized mice, whereas they were significantly suppressed after a 24-h fast. This indicates that the present sampling protocol is suitable for detecting physiological changes in pulsatile LH secretion. In addition, 1-h-interval blood collections in proestrous mice between 1300 and 2200 h revealed that individual preovulatory LH surges occur in the evening of proestrous days. Thus, the present study has developed a blood sampling protocol to detect individual profiles of pulse and surge modes of LH secretion in mice.