A lockstitch is formed by the interlacement of needle and bobbin threads. During high-speed sewing, needle thread is subjected to repeated tensile stresses, heat, bending, pressure, torsion, and wearing. Therefore, the mechanical properties of thread, which become altered during sewing, are very important for its performance and durability. In order to design a sewing machine and threads for maximum strength retention, it is important to understand how the mechanical properties of the thread change as it progresses through the machine and become incorporated into the seam. In this paper, the tensile properties of the needle thread are measured at four sewing stages: before sewing, after dynamic loading at the tension regulator, after passage through the needle and fabric, and after bobbin thread interaction. Mercerized cotton, polyester staple spun, polyester—cotton core spun, and polyester—polyester core spun threads are used and the contribution of dynamic loading, passage through the needle and fabric, and bobbin thread interaction to the change in tensile properties is analyzed. The loss in tenacity, elongation, and breaking energy for cotton thread is the highest, followed by polyester staple and core spun threads. However, the cotton thread shows lower loss in initial modulus as compared with the other threads. Bobbin thread interaction is mainly responsible for the loss in thread strength, elongation, initial modulus, and breaking energy for all threads, barring breaking elongation in cotton threads. Dynamic loading leads to an increase in the initial modulus for all threads. Passage through the needle and fabric assembly has a relatively small contribution to the reduction in tensile properties.