<p>Space exploration is important for scientific discovery, advancing technology, and the long-term survival of humanity. However, the impacts of microgravity and cosmic radiation during space travel on human physiology are not completely understood. While microgravity results in a loss of skeletal muscle mass and function, the effect on cardiac muscle, in particular the contractile elements, is not as clear. Here, we examine the effect of spaceflight on the myocardial contractile function of skinned cardiomyocytes from mice that traveled to the International Space Station (spaceflight, <i>N</i> = 5) and age-matched ground controls (ground control, <i>N</i> = 5, and vivarium, <i>N</i> = 3). These experiments allow for the characterization of the mechanical properties of the sarcomere, the fundamental unit of contraction. The functional experiments showed that ~38.5 days in space does not alter force-generating capacity (T_max), calcium sensitivity (EC₅₀), and cooperativity of the sarcomere. The passive force and cross-sectional area (CSA) were the same between the spaceflight and ground control groups. We next performed mass spectrometry (MS) analysis, and gene ontology analysis confirmed that pathways associated with sarcomere contractility remained unchanged. However, the MS data showed that the spaceflight group exhibited immune‑related proteomic changes compared to the ground controls. Together, these results suggest that ~38.5 days of space travel does not substantially affect the intrinsic contractile state of murine cardiomyocytes.</p>