Background Chronic atrial fibrillation (cAF) is characterized by electric, structural, and contractile remodeling that leads to pronounced atrial contractile dysfunction. To determine the impact of sarcomere alterations on contractile dysfunction in cAF, comparisons were made between membrane-permeabilized cardiomyocytes, muscle strips, and myofibrils isolated from atrial samples of patients in sinus rhythm (SR) and patients with cAF. Measurements of tension development and ATPase acitivity were performed at various calcium concentrations. Results Compared to SR, cAF muscle preparations showed (1) a reduction in maximum tension; (2) a reduction in the rates of tension activation and relaxation; (3) preserved economy of contraction; (4) an increase in myofilament Ca2+-sensitivity; (5) a reduction in myofibril passive tension. The slow β-myosin heavy chain isoform (β-MHC) and the more compliant titin isoform N2BA were up-regulated in cAF compared to SR. Phosphorylation of multiple myofilament proteins was increased in cAF compared to SR atrial myocardium. Conclusions The increased relative amount of the slow β-MHC in cAF directly accounts for the reduction in cross-bridge cycling kinetics of cAF compared to SR muscle preparations. The negative impact of the MHC isoform change on the power output and velocity of atrial contraction may contribute to atrial contractile dysfunction in cAF. The decrease in passive stiffness in cAF myofibrils was entirely explained by a shift in titin isoform composition from the stiff N2B to the compliant N2BA isoform. Alterations in active and passive tension generation at the sarcomere level, explained by translational and post-translational changes of multiple myofilament proteins, are part of the contractile dysfunction of human cAF and may contribute to the self-perpetuation of the arrhythmia and the development of atrial dilatation.