Zebrafish (ZF) provide good models for human cardiac arrhythmias with comparable action potentials and high sequence identity to humans for many ion channels. Previous studies showed that ZF cardiomyocytes spontaneously propagate from embryonic progenitor cells to a mature 3D myocardium in vitro, termed Zebrafish heart aggregates (ZFHAs). Although generated from ZF larvae, ZFHAs show similar cellular architecture to adult ZF in terms of sarcomere structures and cell-to-cell connections. Although, past studies have assessed the contribution of adrenergic stimulation in the electrophysiology of adult and larval ZF, little is known about ZFHAs. This studies aim was to distinguish the role of cardiac adrenergic receptor function in regulating contraction frequency (CF) and inotropic response in larval ZF compared to ZFHAs. To help further validate ZFHAs as an in vitro cardiac model we assessed distinct differences in ECG pattern, CF and size under adrenergic stimulation with phenylephrine. Observations were compared to control ZFHA under normal culture conditions in high glucose DMEM. To generate ZFHAs, whole ZF larvae were homogenized in accordance with schedule 1 methods required by the home office. Cardiac function was observed using an inverted microscope with a high-speed camera. CF was determined by measuring the time interval necessary for 30 heart beats and size of aggregates was measured using ImageJ. Due to lack of consistent morphology in ZFHAs, inotropic patterns were determined using chromogenic software developed in R to analyze differences in perimeter movements. Control ZFHAs show gradual decrease in size and increase in CF between 1-6 days post-homogenization (d.p.h) day1(Size=8191.91083.1, CF=453BPM) day6(Size=7614.61428.3, CF= 6712BMP) while phenylephrine caused increased trends in size and CF compared to control, day1(Size=6252.4, CF=5214BPM) day6(Size=9048.4 CF= 7522BMP). At 13d.p.h, a positive chronotropic change was observed in ZFHAs supplemented with phenylephrine (p=0.0026, n=12), which aligned with a significant increase in size seen at 12d.p.h (p=0.0017, n=12). Finally, phenylephrine caused a significant (p=0.0415, n=74) positive correlation between size and CF (r=0.2376) while control had no correlation between CF and size (p=0.3333, n=253). Chromogenic patterns of ZFHAs in response to phenylephrine showed positive inotropic responses and more defined ECG patterns. This suggests that ZFHA have similar developmental patterns of adrenergic receptors to larval ZF. It is also observed that response to the agonist phenylephrine results in positive chronotropic and inotropic tone that maintains CF and size beyond 6d.p.h. Response of ZFHAs to adrenergic stimulation could highlight important correlations between human and ZFHA response to chronotropic drugs developed for cardiac arrhythmias that focus on adrenergic manipulation.