From birth and throughout their lives, dogs experience a variety of potentially stressful stimuli. Early neurologic stimulation (ENS) is believed to improve the ability of animals to handle stress, however its effects on dogs have not yet been fully explored. This study aimed to evaluate the effects and potential welfare implications of providing ENS to puppies in commercial breeding kennels. Seventy-six puppies, comprising two cohorts in one kennel were studied. Puppies were assigned to one of three treatment groups: ENS, held, or control, and then were marked for identification, and handled daily Monday through Saturday for 21 days, beginning on day three post-partum. ENS puppies received five “Bio Sensor” exercises (Battaglia, 2009). Puppies in the held treatment group were held for 30 seconds, which was the same length of time that was required to apply the Bio Sensor exercises to ENS treated puppies. Control puppies received identification marks daily and health assessments weekly, but otherwise were handled as normal for the breeder’s management plan. To evaluate treatment effects on physical health, all puppies received physical health assessments weekly, and additionally before and after transport to a distributor. To evaluate effects of treatment on behavioral responses to stressors, puppies were assessed shortly before and after transport (a known stressor), using three stranger approach tests and a 3-minute isolation test. Puppies were found to be generally healthy and clean throughout the study. A three-way interaction was observed between treatment, sex, and week of life, which affected puppies’ weights over the first eight weeks of life at the breeder’s kennel prior to the application of stressors (<i>p</i> = 0.006). Female ENS puppies were found to weigh more than their held and control counterparts, while for male puppies, held and control puppies weighed more than ENS puppies. A two-way interaction was observed between treatment and isolation on behavior for a single step of the multi-step stranger approach test performed at the breeder’s kennel (<i>p</i> = 0.025). While more puppies showed affiliative behavioral responses to the experimenter reaching for them after isolation than before, the change was greater in ENS and held treatment groups than controls. Treatment also directly affected the time puppies spent performing fearful behavior during the isolation test (<i>p</i> = 0.041). Handled puppies spent more time performing fearful behaviors than control puppies. No other significant effects of treatment were observed for the behavioral or physical health parameters measured. However, the finding that ENS and held-groups tended to show greater increases in the number of puppies displaying affiliative behavior than controls (though it was only significant for one step) suggests that handling treatments primed puppies to view people as a form of social support during stress. The additional finding that ENS and held group puppies spent more time performing fearful behaviors (e.g., escape attempts, low postures) during isolation than control puppies, further supports this theory. While these results do not support the purported effects of ENS, they indicate that early handling may still benefit puppies by providing them positive interactions with humans. These interactions potentially prime developing puppies to view humans as safe sources of social support, perhaps increasing their likelihood of forming secure attachments with people later in life. Further, findings from this study suggest that simply holding puppies daily for short periods may be sufficient to produce beneficial effects. Future studies should incorporate measures of recovery in response to stress testing puppies receiving ENS treatment and should consider evaluating ENS in conjunction with attachment theory to provide more information on the potential welfare effects of early handling of puppies in commercial breeding and other kennel types.