One the earliest manifestations of cardiac dysfunction in diabetes is diastolic dysfunction, however the exact underlying mechanism is unknown, especially in humans. This study aimed to measure cardiac function in rest and during β-adrenergic stimulation, and also the expression of calcium- cycling proteins and fibrosis in myocardium of diabetic patients without overt systolic function. Right atrial appendages from patients with type 2 diabetes mellitus (DM) and non-diabetic patients (non-DM), all with preserved ejection fraction and undergoing coronary artery bypass grafting (CABG), were collected. Trabeculae, small cardiac muscles, were isolated from the appendages to measure basal and β-adrenergic stimulated myocardial function. Protein expression levels of the calcium-handling proteins, sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) and phospholamban (PLB), and of the 1-adrenoreceptors were determined in tissue samples by Western blot. Collagen deposition as measure of fibrosis was determined by picro-sirius red staining. The contractile function of the trabeculae from diabetic patients was preserved, but its relaxation was prolonged. The protein expression of SERCA2a was increased in diabetic myocardial tissue (0.69 ± 0.06 vs. 1.32 ± 0.25, non-DM vs. DM, p = 0.008), whereas the protein expression of its endogenous inhibitor PLB was reduced (2.51 ± 0.55 vs. 0.66 ± 0.17, non-DM vs. DM, p = 0.02). The collagen deposition was increased in samples from diabetic patients. Moreover, trabeculae from diabetic patients were unresponsive to β-adrenergic stimulation, despite no change in 1 -adrenoreceptor protein expression levels. Although changes in calcium-handling protein expression suggests accelerated active calcium re-uptake (improved relaxation) in human type 2 diabetic atrial myocardium, the functional results showed that contractility was unaltered but relaxation was impaired, especially during β-adrenergic challenge. Thus, the changes in calcium-handling proteins in diabetes might be compensatory in an attempt to maintain diastolic function at rest despite impaired relaxation in the diabetic fibrotic atrial myocardium.