Embryonic Gulf killifish (Fundulus grandis) exhibit precocial parasympathetic-mediated cardiac control, yet little there is little research concerning their unusually early vagal tone. These embryos feature traits uniquely suited for studying early ANS development as 1) they require no anesthesia, thereby eliminating collateral interference that anesthetics have on the ANS or cardiovascular system, 2) feature a semi-transparent chorion, enabling direct observation of the heart, and 3) possess a fully sequenced and annotated genome conducive to understanding genotype-phenotype relationships. Parasympathetic inhibition of the heart created by a ‘startle’ response is evident as a transient cardiac arrest and can be performed by applying gentle mechanical pressure to the embryos under a glass microscope slip, followed by atropine administration upon positive observation of vagal tone. Habituation assays were performed to determine whether an attenuated vagal response to repeated startle stimuli emerged over time, a further expression of early cardiac control. This included obtaining trace data of cardiac activity using a high-throughput heart monitor system for fish embryos. At 80% to hatch a startle stimulus induces transient bradycardia for ~30-40sec. This parasympathetic response was blocked by 1 mmol atropine. Cardiac activity observed during habituation assays showed a non-diminished susceptibility to induced bradycardia. Embryos as young as 60-70% to hatch (~9 days post-fertilization) exhibit vagal tone through parasympathetic stimulation, signifying the initial vagal innervation of the heart. Exploiting this precocial onset of parasympathetic innervation in Gulf killifish embryos can serve as an interface linking established genotypes to relevant physiological phenotypes.