We determined the cardiovascular effects of chronic ₂-adrenoceptor (2AR) stimulation in vivo, and examined the mechanism for the previously observed prolonged diastolic relaxation. Rats (3 months old, n=6), instrumented with implantable radio-telemeters, received the selective 2AR agonist formoterol (25 µg/kg/day, i.p.) for four weeks, with selected cardiovascular parameters measured daily throughout this period, and for a further seven days following cessation of treatment. Chronic 2AR stimulation was associated with an increase in heart rate (HR) of 17% (days 1-14) and 5% (days 15-28), an 11% (D1-14) and 6% (D15-28) decrease in mean arterial blood pressure (MAP), and a 24% (D1-14) increase in the rate of cardiac relaxation (-dP/dt) compared with initial values (P<0.05). Cessation of 2AR stimulation resulted in an 8% decrease in HR and a 7% decrease in -dP/dt, compared with initial values (P<0.05). The prolonged cardiac relaxation following cessation of chronic 2AR stimulation was associated with a ~30% decrease in the maximal rate (V_max) of sarco-/endoplasmic reticulum Ca²⁺-ATPase (SERCA) activity and a ~50% decrease in SERCA2a protein (P<0.05). GSK3 has been implicated as a negative regulator of SERCA2 gene transcription and we observed a ~60% decrease (P<0.05) in phosphorylated GSK3 protein after chronic 2AR stimulation. Finally, we found a 40% decrease (P<0.05) in the mRNA expression of the novel A kinase anchoring protein AKAP18, also implicated in 2AR-mediated cardiac relaxation. These findings highlight some detrimental cardiovascular effects of chronic 2AR agonist administration and identify concerns for their current and future use for treating asthma or for conditions where muscle wasting and weakness are indicated.