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Introduction Dogs are used for various physically demanding sports and work activities, with training starting at a very young age. However, little is known about the effects of training on young puppies. This study investigated effects of a structured endurance program on changes in heart rate (HR) and blood lactate (BL) concentrations measured after a fitness field test in Labrador retriever puppies aged under six months. Methods Fifteen puppies were included: eight in the training group (TRA) and seven in the control group (C). Training group puppies followed an eight-week training program, while C puppies lived their normal lives without any intervention. The amount and intensity of locomotion were monitored continuously. Changes in HR and BL were measured after field tests consisting of a 1,000 m run and a 200 m sprint, followed by a recovery time until HR returned to baseline. Measurements were taken at baseline, mid-timepoint (four weeks of training), end of training (eight weeks of training), and after a detraining period (four weeks after end of training). Results No adverse short-term effects were observed in relation to the training program. Most physiological outcomes were non-significant. After the detraining period, in the TRA-group, HRs were 32 beats per minute lower ( p = 0.040) after the 200 m sprint than at baseline. The only significant change ( p = 0.028) was observed in the BL concentration after the 200 m sprint; after the detraining period, it was lower than after the end-timepoint in the TRA-group. Discussion A safe, periodized training program of varying volume, intensity, and frequency of physical loading was introduced. Limited significant physiological responses were, however, reported. Large variations in HR and BL levels were noted between the puppies throughout the study period. Study's imitations include small sample size, lack of randomization in group allocation, and lack of assessment of long-term impact to the musculoskeletal system.