Objective: To investigate single muscle fiber contractile performance in muscle biopsies from patients with facioscapulohumeral muscular dystrophy, one of the most common hereditary muscle disorders. Methods: we collected 26 vastus lateralis and 24 tibialis anterior muscle biopsies from 14 genetically confirmed FSHD patients and 12 healthy controls. Single muscle fibers (N = 547) were isolated for contractile measurements. Titin content and PEVK phosphorylation were examined in vastus lateralis muscle biopsies. Results: Single muscle fiber specific force is intact at saturating and physiological calcium concentrations in all FSHD biopsies, with (FSHDFAT) and without (FSHDNORMAL) fatty infiltration. Myofilament calcium sensitivity of force is increased in single muscle fibers obtained from FSHD muscle biopsies with increased fatty infiltration, but not in FSHD muscle biopsies without fatty infiltration (pCa50: 5.77-5.80 in healthy control, 5.74-5.83 in FSHDNORMAL and 5.86-5.90 in FSHDFAT single muscle fibers). Cross-bridge cycling kinetics at saturating calcium concentrations and myofilament cooperativity did not differ from healthy controls. Passive force was increased in all FSHD muscle fibers, resulting in increased fiber stiffness. Titin content was increased in FSHD biopsies, however titin phosphorylation did not differ from healthy controls. Conclusion: Muscle weakness in patients with FSHD is not caused by reduced specific force of individual muscle fibers, even in severely affected tissue. Muscle fiber calcium sensitivity of force was increased in severely affected tissue and probably is a compensatory mechanism to maintain specific force. Fiber stiffness was increased in FSHD muscle with and without fatty infiltration, suggesting an early event in disease pathology.