Owner-independent assessments of Diabetes Alert Dog (DAD) behaviour post-placement are currently lacking. Here we describe the first study to simultaneously collect objective DAD behavioural data from CCTV footage and concurrent owner glucose levels via a Flash Glucose Monitoring System (FGMS). Using a pre-defined behavioural ethogram, both trained and non-trained canine behaviours were recorded. Given that dogs are trained to display attention-seeking behaviours when their owners experience fluctuations outside of normal blood glucose levels, we would expect differences in DAD behaviour during periods when owner glucose levels transition from euglycaemia to hyperglycaemic (high) or hypoglycaemic (low) levels, as compared to periods when owners stay within target-range. A FreeStyle Libre FGMS was given to nine owners of accredited DADs from a single training establishment. Behavioural data were collected using CCTV in the participants' home, or place of work, for between five and 14 days (mean = 12.2 days). For each person, between 19 and 29 (mean = 22.5) one-hour periods were selected that captured approximately equal instances of owner glucose levels transitioning from in-range to hypoglycaemia, in-range to hyperglycaemia, or remaining within target-range. Two researchers coded footage without knowledge of the owner's glucose levels. Variables recorded included the DAD's Activity, Attentional State, Proximity to Owner, Attention-seeking, and potential Stress-related behaviours. There were significant differences between individual dogs' behaviour during in-range periods. When samples captured a transition to out-of-range glucose levels (hypo- or hyperglycaemia), the distribution of several behaviours among dogs in the cohort differed significantly from their distributions during in-range samples (p < 0.01 for Conover Tests in Playing with Owner, Jump Up, Sniff Owner, Bark, Paw Owner, and Lick Owner), but consistent increases or decreases in the rate of any behaviours were not detected across the cohort. In individual dogs, we found distinctive behaviour changes during periods when their owner's glucose transitioned to out-of-range, as compared to when they remained in-range. Each DAD showed significant changes in the variance of at least one trained attention-seeking behaviour, with several dogs also showing changes in non-trained behaviours such as Change of State, Playing with Owner and potential stress-related behaviours (Yawning and Lip-Licking). This is the first study to objectively show that DADs differ in their behaviour during periods of owner glucose fluctuation and further highlights the individuality of responses. Understanding this variation, and factors affecting it, is fundamental to optimising DAD performance.