The rail fastening system plays a crucial role in railway tracks as it ensures operational safety by fixing the rail on to the sleeper. Early detection of rail fastener system defects is crucial to ensure track safety and to enable maintenance optimization. Fastener inspections are normally conducted either manually by trained maintenance personnel or by using automated 2-D visual inspection methods. Such methods have drawbacks when visibility is limited, and they are also found to be expensive in terms of system maintenance cost and track possession time. In a previous study, the authors proposed a train-based differential eddy current sensor system based on the principle of electromagnetic induction for fastener inspection that could overcome the challenges mentioned above. The detection in the previous study was carried out with the aid of a supervised machine learning algorithm. This study reports the finding of a case study, along a heavy haul line in the north of Sweden, using the same eddy current sensor system mounted on an in-service freight train. In this study, unsupervised machine learning models for detecting and analyzing missing clamps in a fastener system were developed. The differential eddy current measurement system was set to use a driving field frequency of 27 kHz. An anomaly detection model combining isolation forest (IF) and connectivity-based outlier factor (COF) was implemented to detect anomalies from fastener inspection measurements. To group the anomalies into meaningful clusters and to detect missing clamps within the fastening system, an unsupervised clustering based on the DBSCAN algorithm was also implemented. The models were verified by measuring a section of the track for which the track conditions were known. The proposed anomaly detection model had a detection accuracy of 96.79% and also exhibited a high score of sensitivity and specificity. The DBSCAN model was successful in clustering missing clamps, both one and two missing clamps, from a fastening system separately.