Tire coming-off accident of large vehicles has developed into a social problem that threatens the safety of civic life. One of the major causes of the accidents was that the tire tightening operation had usually been conducted by impact wrenches without torque control function. It is strongly recommended since 2003 to use torque wrenches especially designed for wheel bolts of large vehicles. However, it has not yet been achieved to drastically reduce the number of tire-coming off accidents. Most accidents are traced to the fatigue failure of wheel bolts that tighten the tire wheels to the hub. Therefore, it is strongly desired to elucidate the fatigue failure mechanism of wheel bolts. In this paper, stress amplitudes generated in wheel bolts during tire rotation are measured under live load. The test equipment is constructed utilizing an actual trailer. Compression loads exerted on the wheel bolts are varied corresponding to the actual loading capacity of the trailer including the overloading state. The magnitudes of bolt clamping forces are also changed so as to cover from the normal clamping condition to the loosening one where the wheel bolts are tightened with insufficient clamping forces. Experimental results show that insufficient clamping forces, e.g., less than 30% of the normal value, cause high stress amplitudes in wheel bolts.