Cardiovascular diseases (CVDs) are the Number-1 killer worldwide with over 17.5 million deaths per year1, and the use of preclinical murine models has greatly aided such understanding. Live, real-time, in-vivo ultrasound imaging is one great tool for studying as dynamic an organ as the heart and is especially successful for small animals. However, preclinical studies rely on imaging under anaesthesia due to difficulties in compliance of the animals to stay still, which has several limitations: 1) It is not a true physiological representation of the disease, potentially modifying the therapeutic-reponse, and measurements are likely to be confounded by effects of anaethesia; 2) With numerous studies documenting the cardio-depressive effects of anaethesia2,3, it is especially not suitable for studies of the heart; 3) Clinically, patients are imaged in the ‘awake’ (aka conscious) state without the use of anaethetics, hence the difference in method does not allow horizontal-comparison of results and often leads to promising preclinical research not translated into the clinic. Here we have developed a clinically-relevant preclinical method of measuring cardiac function in ‘awake’ and ‘slept’ mice using real-time cardiac ultrasound. We aim to inform researchers on the various differences in cardiac function between animals imaged under anaesthesia and in the ‘awake’ state, and hope to propose a new regime for studying cardiovascular function via echocardiography in the preclinical setting. Ten female BALB/c mice were shaved one-day prior (-1d) to echocardiography and were consecutively imaged on a daily basis (1d to 5d), first in the ‘awake’ state (securely- but gently-scruffed) and then in the ‘slept’ state (anaesthetised, 2% isoflurane in 100% oxygen). Parasternal long- and short-axis views in B- and M-modes were obtained. Ejection fraction (EF) was significantly higher in awake mice and showed less variability at all time-points compared to anaesthetised (96.0±5.3 vs 47.9±9.6%). Stroke volume (SV) initially-similar, but reduced overtime (22.8±6.7 vs 27.7±4.4μl at 1d; 9.5±4.7 vs 25.1±6.6μl at 5d). Cardiac output (CO) was comparable (12.2±5.5 vs 11.6±2.5ml/min). In conclusion, we have developed a clinically-relevant preclinical method of measuring cardiac function and showed the feasibility of imaging conscious mice using real-time cardiac ultrasound. EF was higher in ‘awake’ state, as expected without the cardiac-depressive-effects of anaesthesia, thereby allowing greater-sensitivity in detecting changes after therapy/treatment. Variability was also reduced, possibly due to the absence of sinus rhythm-disturbance caused by anaesthesia, thus increasing the chances of observing a true effect. SV reduced overtime, possibility-reflecting training of the animals to the procedure. CO was similar between the two states. Taken all together, we propose this new method of imaging to better-align preclinical to clinical ultrasound imaging of cardiac function.