The effect of three-layer weft knitted spacer material content and structure upon the characteristics of uniaxial tension, biaxial punching before and after fatigue punching, and at multi cycle low-stress (wearing level) punching were investigated. Three different spacer structures were knitted using four types of the same 20 tex linear density spun yarns. For three-layer weft knitted samples, innovative fibers, which are used in the production of functional clothing, were selected: Coolplus® 100% polyester (PES); Trevira CS® 100% PES; 60% PES; 40%PES, 60% PES with 0.02% carbon fiber and 67% cotton, 33% PES with 0.02% carbon fiber. It was defined that the samples, which have a plain jersey knitting pattern in the face and back layers, and a similar position of spacer yarns on the dial and cylinder needles, have the highest values of mass per unit area, but 1 × 1 tuck stitches in the face layer have a greater effect for material thickness. Besides, 1 × 1 tuck stitches in the face layer have a greater effect upon the uniaxial behavior and anisotropy of three-layer weft knitted spacer materials than on spacer yarn position in the connecting layer. The investigations of biaxial fatigue punching up to 50 N, which corresponds to wearing conditions, showed that the most stable and less deformable materials were the three-layer weft knitted spacer materials with a plain jersey knitting pattern in the face and back layers and a similar position of spacer yarns on the dial and cylinder needles. They also were characterized by the highest mass per unit area and medium thickness.