**README** **Peaks 3 'Spring'** Update from March 2023. The zip file contains an updated version of Peaks with following additions: - Ability to select a time window within the recording for which detection is done. Useful when you want to analyse only a section of a very long, gap-free recording. In the Setting window, click the button 'Time Window' and select a starting and end point on your trace, and then press Enter on your keyboard. The time points will then be added to the new text boxes in the Settings window. - Inclusion of both a compiled Windows and macOS version, meaning you do not need to have Matlab installed on your computer. Check the relevant folders for instructions on installing the Matlab Runtime Environment and the application. **Peaks 3 'Purple'** *System Requirements* This software has been developed and tested with Matlab 2017b on macOS 10.14.6, though it is known to work on earlier versions of Matlab and macOS too. No non-standard hardware is required to use the software. Several Matlab toolboxes are used by the software: Signal Processing Toolbox, Image Processing Toolbox, DSP System Toolbox and Curve Fitting Toolbox. *Installation* Unpack the provided zip file and place the peaks folder within the Matlab path. This should be done in under one minute. *Instructions for Use* The software is started by executing the peaks3.m script. The distributed software comes with validation data in the .abf format. The standard settings for detecting APs will result in valid results using these settings. Other data formats that can be loaded are .mat files originating from the HEKA Patchmaster software, .csv and .txt files from any source, including fluorescence traces exported from Fiji. On a normal, current computer loading typical data should be a matter of seconds, or less. For averaging complexes, e.g. electrocardiogram signals, a separate script is included, average_complex_ecg.m. After starting this script and loading a text file containing the data, complexes are found automatically with adjustable sensitivity, and are manually selected. First step is to define a time window for averaging, by selecting the start, peak and end of one specific complex. Next, other complexes are selected to be added to the set for averaging. Selecting 'Align' calculates and plots the average complex +/- SD, which is saved as a text file after pressing 'Save'. This averaged complex may be subsequently analysed using the peaks3.m script. *Expected Output* Output generated by Peaks includes a description of each peak (i.e. AP or calcium transient) detected. Parameters analysed are upstroke velocity, upstroke time, action potential duration at various percentages of repolarisation and peak amplitude and baseline. In addition, beat to previous beat STV is calculated, which after averaging fro 30 beats can be used to calculate short-term variation of repolarisation. *How to run the software on your data* Loading .abf or .mat files from Patchmaster is trivial. For text-based files, the software assumes that the first column contains time, or frame number, whereas the following columns contain data for each channel. Upon loading a text file, the user is asked to confirm the sample time step, default is 100 ms. Up to 4 channels can be displayed.