The adoptability of an animal from a shelter largely depends upon its socialization and friendliness towards humans. For kittens, habituation and proper socialization is an important part of ensuring that the adult cat it will be able to interact properly with humans, thus reducing its chance of being relinquished in the future. In addition, kittens that have been relinquished or placed into a shelter are often subject to several stressors that may impact not only the well-being of the kitten but impair further socialization attempts. The objective of this study was to determine if there were behavioral differences between two handling regimes for kittens relinquished to the ARL-IA. This study was conducted at the Animal Rescue League of Iowa (ARLIA), and involved 31 neonate kittens of mixed sex and breed, between 6 and 8 weeks of age. Treatment (5-d) five consecutive days of handling and treatment two (10-d) ten consecutive days of handling. During treatment kittens were exposed to several tests. The experimental unit was the kitten and a complete randomized experimental design was utilized. PROC GLIMMIX was used. A P-value of P ≤ 0.05 was considered significant. There were differences (P < 0.05) between treatments for the amount of time that kittens were willing to let their rear paws be held. Kittens assigned to 5-d allowed both their rear paws to be held longer than those from the 10-d treatment. For all other handling measures there were no differences. In conclusion, handling kittens over 10-consectuive days indicated that kittens became less tolerant of having their rear paws held compared to a 5-d treatment and therefore there may be an optimal amount of handling before kittens begin to find this procedure more aversive.