Throughout their lives, dogs may experience various stressful events. Early neurological stimulation (ENS), which was shown to alter stress responses beneficially in some animals, has not been fully explored in dogs. Seventy-six small-breed puppies from one commercial breeding kennel were divided into three treatment groups: ENS, held, and control. Puppies in the ENS group received 30 s of handling exercises for 21 days after birth; puppies in the held group were simply held for the same amount of time. Puppies in the control group were managed as per normal breeder practices (i.e., routine husbandry and physical health checks). Physical health was assessed weekly, and puppies were generally healthy and clean. Behavioral responses to stranger approach and isolation tests were evaluated pre- and post-ground-transportation to a distributor. Puppies were more affiliative toward a stranger post-isolation than pre-isolation (p < 0.001), and post-transport than pre-transport (p < 0.001). At the distributor, puppies in the isolation test spent less time in exploratory locomotion (p < 0.001) and vocalized more than at the breeder’s kennel (p = 0.011). Treatment did not affect these results. Overall, the results suggest that the type of ENS used in this study may not provide the purported benefits to puppies’ stress responses in commercial breeding populations.