Negative CLO test pellets can be reused repeatedly in diagnostic endoscopy within a short period of time. However, the duration that these pellets can be stored at room temperature before second use remains unclear. A total of 360 patients, 190 males and 170 females, who required a CLO test during endoscopy, were enrolled in this study. Two biopsies were taken from the gastric antrum of each patient, one for testing with a new pellet and the other for testing with a reused pellet. The reused pellets were used randomly and were divided into five groups according to the time interval between their initial and second usages (1, 2, 3, 6, and > 6 mo). When a positive result was found, the time to color change was recorded. Good correlation was noted for nearly all the paired CLO tests in all groups with either both positive or both negative. Only four pairs produced discrepant results. There was no significant difference when the results of both new and reused CLO tests were compared using McNemar's test (p > 0.05). In positive pairs, there was no significant difference in the color change time of both tests in all five groups by two-tailed t-test (p > 0.05); Pearson's correlation and linear regression showed a strong correlation between the color time change in the five groups (p < 0.0001). Only 54 of the 427 negative pellets stored for more than 6 months could be reused because most were dried out or no longer yellow in color. In conclusion, negative CLO test pellets may be reused within 6 months after initial usage provided they are stored at room temperature.