Tree root respiration is an important component of the carbon balance in forest ecosystems; however, it is not clear whether root preparation treatments (such as excising and washing) affect root respiration measurements. Here, we aimed to compare the respiration rates of roots subjected to different treatments (i.e., washing with water vs. brushing without water, and excising vs. not excising) for 17-month-old seedlings of Cryptomeria japonica. Immediately after sampling an entire root system, the root respiration rate was measured on a mass basis using a closed static chamber system equipped with an infrared gas analyzer. We found that the respiration rates for roots that were excised 10–20 times were significantly higher than those for roots that were not excised. There was no significant difference in the root respiration rates between washing and brushing treatments. Our results indicate that large numbers of excisions (>10 times) could lead to bias in the measured changes in specific root respiration rates, and imply that differences between washing and brushing treatments do not affect the specific root respiration rate. We conclude that potential variation in recorded root respiration rates could be minimized by standardizing the root preparation technique, which should involve rapidly removing all loose soil and limiting the extent of root excision.