We previously reported a series of macrocyclic analogues of [Pyr1]-apelin-13 (Ape13) with increased plasma stability and potent APJ agonist properties. Based on the most promising compound in this series, we synthesized and then evaluated novel macrocyclic compounds of Ape13 to identify agonists with specific pharmacological profiles. These efforts led to the development of analogues 39 and 40, which possess reduced molecular weight (MW 1020 Da vs Ape13, 1534 Da). Interestingly, compound 39 (Ki 0.6 nM), which does not activate the Gα12 signaling pathway while maintaining potency and efficacy similar to Ape13 to activate Gαi1 (EC50 0.8 nM) and β-arrestin2 recruitment (EC50 31 nM), still exerts cardiac actions. In addition, analogue 40 (Ki 5.6 nM), exhibiting a favorable Gα12-biased signaling and an increased in vivo half-life (t1/2 3.7 h vs <1 min of Ape13), produces a sustained cardiac response up to 6 h after a single subcutaneous bolus injection.